Vaccine

ABSTRACT

The present invention relates to the field of bacterial polysaccharide antigen vaccines. In particular, the present invention relates to vaccines comprising a pneumococcal polysaccharide antigen, typically a pneumococcal polysaccharide conjugate antigen, formulated with a protein antigen form  Streptococcus pneumoniae , and optionally a Th1-inducing adjuvant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. Ser. No. 11/216,226filed 31 August 20005, which is a continuation of Ser. No. 09/936,985filed 19 Dec. 2001 (abandoned), which is a 371 of PCT/EP00/02467 filed17 Mar. 2000 (abandoned) (the contents of each are incorporated hereinby reference in their entirety). This application also claims priorityto Great Britain applications GB99/06437 filed on 19 Mar. 1999,GB99/09077.1 filed on 20 Apr. 1999, GB99/09466.6 filed 23 Apr. 1999 andGB99/16677.9 filed on 15 Jul. 1999.

FIELD OF INVENTION

The present invention relates to bacterial polysaccharide antigenvaccines, their manufacture and the use of such polysaccharides inmedicines.

In particular the present invention relates to three inter-relatedaspects: A—vaccines comprising a pneumococcal polysaccharide antigen,typically a pneumococcal polysaccharide conjugate antigen, formulatedwith a protein antigen from Streptococcus pneumoniae and optionally aTh1 inducing adjuvant; B—specific, advantageous pneumococcalpolysaccharide conjugates adjuvanted with a Th1 adjuvant; andC—bacterial polysaccharide conjugates in general conjugated to protein Dfrom H. influenzae.

BACKGROUND OF INVENTION

Streptococcus pneumoniae is a Gram-positive bacteria responsible forconsiderable morbidity and mortality (particularly in the young andaged), causing invasive diseases such as pneumonia, bacteremia andmeningitis, and diseases associated with colonisation, such as acuteOtitis media. The rate of pneumococcal pneumonia in the US for personsover 60 years of age is estimated to be 3 to 8 per 100,000. In 20% ofcases this leads to bacteremia, and other manifestations such asmeningitis, with a mortality rate close to 30% even with antibiotictreatment.

Pneumococcus is encapsulated with a chemically linked polysaccharidewhich confers serotype specificity. There are 90 known serotypes ofpneumococci, and the capsule is the principle virulence determinant forpneumococci, as the capsule not only protects the inner surface of thebacteria from complement, but is itself poorly immunogenic.Polysaccharides are T-independent antigens, and can not be processed orpresented on MHC molecules to interact with T-cells. They can however,stimulate the immune system through an alternate mechanism whichinvolves cross-linking of surface receptors on B cells.

It was shown in several experiments that protection against invasivepneumococci disease is correlated most strongly with antibody specificfor the capsule, and the protection is serotype specific.

Polysaccharide antigen based vaccines are well known in the art. Fourthat have been licensed for human use include the Vi polysaccharide ofSalmonella typhi, the PRP polysaccharide from Haemophilus influenzae,the tetravalent meningococcal vaccine composed of serotypes A, C, W135and Y, and the 23-Valent pneumococcal vaccine composed of thepolysaccharides corresponding to serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N,9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33(accounting for at least 90% of pneumococcal blood isolates).

The latter three vaccines confer protection against bacteria causingrespiratory infections resulting in severe morbidity and mortality ininfants, yet these vaccines have not been licensed for use in childrenless than two years of age because they are inadequately immunogenic inthis age group [Peltola et al., (1984), N. Engl. J. Med. 310:1561-1566].Streptococcus pneumoniae is the most common cause of invasive bacterialdisease and otitis media in infants and young children. Likewise, theelderly mount poor responses to pneumococcal vaccines [Roghmann et al.,(1987), J. Gerontol. 42:265-270], hence the increased incidence ofbacterial pneumonia in this population [Verghese and Berk, (1983)Medicine (Baltimore) 62:271-285].

Strategies, which have been designed to overcome this lack ofimmunogenicity in infants, include the linking of the polysaccharide tolarge immunogenic proteins, which provide bystander T-cell help andwhich induce immunological memory against the polysaccharide antigen towhich it is conjugated. Pneumococcal glycoprotein conjugate vaccines arecurrently being evaluated for safety, immunogenicity and efficacy invarious age groups.

A) Pneumococcal Polysaccharide Vaccines

The 23-valent unconjugated pneumococcal vaccine has shown a widevariation in clinical efficacy, from 0% to 81% (Fedson et al. (1994)Arch Intern Med. 154: 2531-2535). The efficacy appears to be related tothe risk group that is being immunised, such as the elderly, Hodgkin'sdisease, splenectomy, sickle cell disease and agammaglobulinemics (Fineet al. (1994) Arch Intern Med. 154:2666-2677), and also to the diseasemanifestation. The 23-valent vaccine does not demonstrate protectionagainst pneumococcal pneumonia (in certain high risk groups such as theelderly) and otitis media diseases.

There is therefore a need for improved pneumococcal vaccinecompositions, particularly ones which will be more effective in theprevention or amelioration of pneumococcal disease (particularlypneumonia) in the elderly and in young children.

The present invention provides such an improved vaccine.

B) Selected Pneumococcal Polysaccharide Conjugate+3D-MPL Compositions

It is generally accepted that the protective efficacy of thecommercialised unconjugated pneumococcal vaccine is more or less relatedto the concentration of antibody induced upon vaccination; indeed, the23 polysaccharides were accepted for licensure solely upon theimmunogenicity of each component polysaccharide (Ed. Williams et al. NewYork Academy of Sciences 1995 pp. 241-249). Therefore furtherenhancement of antibody responses to the pneumococcal polysaccharidescould increase the percentage of infants and elderly responding withprotective levels of antibody to the first injection of polysaccharideor polysaccharide conjugate and could reduce the dosage and the numberof injections required to induce protective immunity to infectionscaused by Streptococcus pneumoniae.

Since the early 20^(th) century, researchers have experimented with ahuge number of compounds which can be added to antigens to improve theirimmunogenicity in vaccine compositions [reviewed in M. F. Powell & M. J.Newman, Plenum Press, NY, “Vaccine Design—the Subunit and AdjuvantApproach” (1995) Chapter 7 “A Compendium of Vaccine Adjuvants andExcipients”]. Many are very efficient, but cause significant local andsystemic adverse reactions that preclude their use in human vaccinecompositions. Aluminium-based adjuvants (such as alum, aluminiumhydroxide or aluminium phosphate), first described in 1926, remain theonly immunologic adjuvants used in human vaccines licensed in the UnitedStates.

Aluminium-based adjuvants are examples of the carrier class of adjuvantwhich works through the “depot effect” it induces. Antigen is adsorbedonto its surface and when the composition is injected the adjuvant andantigen do not immediately dissipate in the blood stream—instead thecomposition persists in the local environment of the injection and amore pronounced immune response results. Such carrier adjuvants have theadditional known advantage of being suitable for stabilising antigensthat are prone to breakdown, for instance some polysaccharide antigens.

3D-MPL is an example of a non-carrier adjuvant. Its full name is3-O-deacylated monophosphoryl lipid A (or 3 De-O-acylated monophosphoryllipid A or 3-O-desacyl-4′ monophosphoryl lipid A) and is referred to as3D-MPL to indicate that position 3 of the reducing end glucosamine isde-O-acylated. For its preparation, see GB 2220211 A. Chemically it is amixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylatedchains. It was originally made in the early 1990's when the method to3-O-deacylate the 4′-monophosphoryl derivative of lipid A (MPL) led to amolecule with further attenuated toxicity with no change in theimmunostimulating activity.

3D-MPL has been used as an adjuvant either on its own or,preferentially, combined with a depot-type carrier adjuvant such asaluminium hydroxide, aluminium phosphate or oil-in-water emulsions. Insuch compositions antigen and 3D-MPL are contained in the sameparticulate structures, allowing for more efficient delivery ofantigenic and immunostimulatory signals. Studies have shown that 3D-MPLis able to further enhance the immunogenicity of an alum-adsorbedantigen [Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1]. Suchcombinations are also preferred in the art for antigens that are proneto adsorption (for instance, bacterial polysaccharide conjugates), whereadsorption onto alum tends to stabilise the antigen. Precipitatedaluminium-based adjuvants are mostly used as they are the only adjuvantsthat are currently used in licensed human vaccines. Accordingly,vaccines containing 3D-MPL in combination with aluminium-based adjuvantsare favoured in the art due to their ease of development and speed ofintroduction onto the market.

MPL (non 3-deacylated) has been evaluated as an adjuvant with severalmonovalent polysaccharide-conjugate vaccine antigens. Coinjection of MPLin saline enhanced the serum antibody response for four monovalentpolysaccharide conjugates: pneumococcal PS 6B-tetanus toxoid,pneumococcal PS 12-diphtheria toxoid, and S. aureus type 5 and S. aureustype 8 conjugated to Pseudomonas aeruginosa exotoxin A [Schneerson etal. J. Immunology (1991) 147:2136-2140]. The enhanced responses weretaught as being antigen-specific. MPL in an oil-in-water emulsion (acarrier type adjuvant) consistently enhanced the effect of MPL in salinedue to the presence of MPL and antigen in the same particulatestructure, and was considered to be the adjuvant system of choice foroptimal delivery of other polysaccharide conjugate vaccines.

Devi et al. [Infect. Immun. (1991) 59:3700-7] evaluated the adjuvanteffect of MPL (non 3-deacylated) in saline on the murine antibodyresponse to a TT conjugate of Cryptococcus neoformans capsularpolysaccharide. When MPL was used concurrently with the conjugate therewas only a marginal increase in both the IgM- and IgG-specific responseto the PS; however MPL had a much larger effect when administered 2 daysafter the conjugate. The practicality of using an immunization schemethat requires a delay in the administration of MPL relative to antigen,especially in infants, is questionable. The adjuvant effect of MPL withpolysaccharides and polysaccharide-protein conjugates appears to becomposition-dependent. Again, the incorporation of MPL in a suitableslow-release delivery systems (for instance using a carrier adjuvant)provides a more durable adjuvant effect and circumvents the problem oftiming and delayed administration.

In summary, the state of the art has taught that, for particularpolysaccharide or polysaccharide-conjugate antigens, where MPL or 3D-MPLis used as an adjuvant, it is advantageously used in conjuction with acarrier adjuvant (for instance the aluminium-based adjuvants) in orderto maximise its immunostimulatory effect.

Surprisingly, the present inventors have found that for certainpneumococcal polysaccharide conjugates, the immunogenicity of thevaccine composition is significantly greater when the antigen isformulated with 3D-MPL alone rather than with 3D-MPL in conjunction witha carrier adjuvant (such as an aluminium-based adjuvant). Furthermorethe observed improvement is independent of the concentration of 3D-MPLused, and whether the particular conjugates are in a monovalentcomposition or whether they are combined to form a polyvalentcomposition.

C) Bacterial Polysaccharide—Protein D Conjugates

As mentioned above, polysaccharide antigen based vaccines are well knownin the art. The licensed polysaccharide vaccines mentioned above havedifferent demonstrated clinical efficacy. The Vi polysaccharide vaccinehas been estimated to have an efficacy between 55% and 77% in preventingculture confirmed typhoid fever (Plotkin and Cam, (1995) Arch Intern Med155: 2293-99). The meningococcal C polysaccharide vaccine was shown tohave an efficacy of 79% under epidemic conditions (De Wals P, et al.(1996) Bull World Health Organ. 74: 407-411). The 23-valent pneumococcalvaccine has shown a wide variation in clinical efficacy, from 0% to 81%(Fedson et al. (1994) Arch Intern Med. 154: 2531-2535). As mentionedabove, it is accepted that the protective efficacy of the pneumococcalvaccine is more or less related to the concentration of antibody inducedupon vaccination.

Amongst the problems associated with the polysaccharide approach tovaccination, is the fact that polysaccharides per se are poorimmunogens. Strategies which have been designed to overcome this lack ofimmunogenicity include the linking of the polysaccharide to large highlyimmunogenic protein carriers, which provide bystander T-cell help.

Examples of these highly immunogenic carriers which are currentlycommonly used for the production of polysaccharide immunogens includethe Diphtheria toxoid (DT or the CRM197 mutant), Tetanus toxoid (TT),Keyhole Limpet Haemocyanin (KLH), and the purified protein derivative ofTuberculin (PPD).

Problems Associated with Commonly-Used Carriers

A number of problems are associated with each of these commonly usedcarriers, including in production of GMP conjugates and also inimmunological characteristics of the conjugates.

Despite the common use of these carriers and their success in theinduction of anti polysaccharide antibody responses they are associatedwith several drawbacks. For example, it is known that antigen specificimmune responses may be suppressed (epitope suppression) by the presenceof preexisting antibodies directed against the carrier, in this caseTetanus toxin (Di John et al; (1989) Lancet, 2:1415-8). In thepopulation at large, a very high percentage of people will havepre-existing immunity to both DT and TT as people are routinelyvaccinated with these antigens. In the UK for example 95% of childrenreceive the DTP vaccine comprising both DT and TT. Other authors havedescribed the problem of epitope suppression to peptide vaccines inanimal models (Sad et al, Immunology, 1991; 74:223-227; Schutze et al,J. Immunol. 135: 4, 1985; 2319-2322).

In addition, for vaccines which require regular boosting, the use ofhighly immunogenic carriers such as TT and DT are likely to suppress thepolysaccharide antibody response after several injections. Thesemultiple vaccinations may also be accompanied by undesirable reactionssuch as delayed type hyperresponsiveness (DTH).

KLH is known as potent immunogen and has already been used as a carrierfor IgE peptides in human clinical trials. However, some adversereactions (DTH-like reactions or IgE sensitisation) as well as antibodyresponses against antibody have been observed.

The selection of a carrier protein, therefore, for a polysaccharidebased vaccine will require a balance between the necessity to use acarrier working in all patients (broad MHC recognition), the inductionof high levels of anti-polysaccharide antibody responses and lowantibody response against the carrier.

The carriers used previously for polysaccharide based vaccines,therefore have many disadvantages. This is particularly so incombination vaccines, where epitope suppression is especiallyproblematic if the same carrier is used for various polysaccharideantigens. In WO 98/51339, multiple carriers in combination vaccines wereused in order to try to get over this effect.

The present invention provides a new carrier for use in the preparationof polysaccharide/polypeptide-based immunogenic conjugates, that doesnot suffer from the aforementioned disadvantages.

The present invention provides a protein D (EP 0 594 610 B1) fromHaemophilus influenzae, or fragments thereof, as a carrier forpolysaccharide based immunogenic compositions, including vaccines. Theuse of this carrier is particularly advantageous in combinationvaccines.

SUMMARY OF THE INVENTION A) Pneumococcal Polysaccharide Vaccines

Accordingly the present invention provides a vaccine composition,comprising at least one Streptococcus pneumoniae polysaccharide antigen(preferably conjugated) and a Streptococcus pneumoniae protein antigenor immunologically functional equivalent thereof, optionally with a Th1adjuvant (an adjuvant inducing a Th1 immune response). Preferably both apneumococcal protein and Th1 adjuvant are included. The compositions ofthe invention are particularly suited in the treatment of elderlypneumonia.

Pneumococcal polysaccharide vaccines (conjugated or not) may not be ableto protect against pneumonia in the elderly population for which theincidence of this disease is very high. The key defense mechanismagainst the pneumococcus is opsonophagocytosis (a humoralB-cell/neutrophil mediated event caused by the production of antibodiesagainst the pneumococcal polysaccharide, the bacterium eventuallybecoming phagocytosed), however parts of the involved opsonic mechanismsare impaired in the elderly, i.e. superoxide production by PMN(polymorphonuclear cells), other reactive oxygen species production,mobilization of PMN, apoptosis of PMN, deformability of PMN. Antibodyresponses may also be impaired in the elderly.

Contrary to the normally accepted dogma, normal levels of anti-capsularpolysaccharide antibodies may not be effective in complete clearance ofbacteria, as pneumococci may invade host cells to evade this branch ofthe immune system.

Surprisingly, the present inventors have found that by simultaneouslystimulating the cell mediated branch of the immune system (for instanceT-cell meditated immunity) in addition to the humoral brach of theimmune system (B-cell mediated), a synergy (or cooperation) resultswhich is capable of enhancing the clearance of pneumococci from thehost. This is a discovery which will aid the prevention (or treatment)of pneumococcal infection in general, but will be particularly importantfor the prevention (or treatment) of pneumonia in the elderly wherepolysaccharide based vaccines do not show efficacy.

The present inventors have found that both arms of the immune system maysynergise in this way if a pneumococcal polysaccharide (preferablyconjugated) is administered with a pneumococcal protein (preferably aprotein expressed on the surface of pneumococci, or secreted orreleased, which can be processed and presented in the context of ClassII and MHC class I on the surface of infected mammalian cells). Althougha pneumococcal protein can trigger cell mediated immunity by itself, theinventors have also found that the presence of a Th1 inducing adjuvantin the vaccine formulation helps this arm of the immune system, andsurprisingly further enhances the synergy between both arms of theimmune system.

B) Selected Pneumococcal Polysaccharide Conjugate+3D-MPL Compositions

Accordingly, the present invention also provides an antigeniccomposition comprising one or more pneumococcal polysaccharideconjugates adjuvanted with 3D-MPL and substantially devoid ofaluminium-based adjuvants, wherein at least one of the pneumococcalpolysaccharide conjugates is significantly more immunogenic incompositions comprising 3D-MPL in comparison with compositionscomprising 3D-MPL in conjunction with an aluminium-based adjuvant.

Preferred embodiments provided are antigenic compositions comprisingconjugates of one or more of the following pneumococcal capsularpolysaccharides: serotype 4, 6B, 18C, 19F, and 23F. In suchcompositions, each of the polysaccharides are surprisingly moreimmunogenic in compositions comprising 3D-MPL alone compared withcompositions comprising 3D-MPL and an aluminium-based adjuvant.

Thus is one embodiment of the invention there is provided a antigeniccomposition comprising the Streptococcus pneumoniae capsularpolysaccharide serotype 4, 6B, 18C, 19F or 23F conjugated to animmunogenic protein and 3D-MPL adjuvant, wherein the composition issubstantially devoid of aluminium-based adjuvants.

In a second embodiment, the present invention provides a combinationantigenic composition substantially devoid of aluminium-based adjuvantsand comprising 3D-MPL adjuvant and two or more pneumococcalpolysaccharide conjugates chosen from the group consisting of: serotype4; serotype 6B; serotype 18C; serotype 19F; and serotype 23F.

C) Bacterial Polysaccharide—Protein D Conjugates

Accordingly, the present invention provides a polysaccharide conjugateantigen comprising a polysaccharide antigen derived from a pathogenicbacterium conjugated to protein D from Haemophilus influenzae or aprotein D fragment thereof. In addition, the invention providespolyvalent vaccine compositions where one or more of the polysaccharideantigens are conjugated to protein D.

DESCRIPTION OF THE INVENTION A) Pneumococcal Polysaccharide Vaccines

The present invention provides an improved vaccine particularly for theprevention or amelioration of pnemococcal infection of the elderly(and/or infants and toddlers).

In the context of the invention a patient is considered elderly if theyare 55 years or over in age, typically over 60 years and more generallyover 65 years.

Thus in one embodiment of the invention there is provided a vaccinecomposition, suitable for use in the elderly (and/or Infants andtoddlers) comprising at least one Streptococcus pneumoniaepolysaccharide antigen and at least one Streptococcus pneumoniae proteinantigen.

In a second, preferred, embodiment, the present invention provides avaccine (suitable for the prevention of pneumonia in the elderly)comprising at least one Streptococcus pneumoniae polysaccharide antigenand at least one Streptococcus pneumoniae protein antigen and a Th1adjuvant.

It is envisaged that such a vaccine will be also useful in treatingpneumococcal infection (for instance otitis media) in other high riskgroups of the population, such as for infants or toddlers.

In a third embodiment there is provided a vaccine composition comprisinga pneumococcal polysaccharide antigen and a Th1 adjuvant.

Streptococcus pneumoniae Polysaccharide Antigens of the Invention

Typically the Streptococcus pneumoniae vaccine of the present inventionwill comprise polysaccharide antigens (preferably conjugated), whereinthe polysaccharides are derived from at least four serotypes ofpneumococcus. Preferably the four serotypes include 6B, 14, 19F and 23F.More preferably, at least 7 serotypes are included in the composition,for example those derived from serotypes 4, 6B, 9V, 14, 18C, 19F, and23F. More preferably still, at least 11 serotypes are included in thecomposition, for example the composition in one embodiment includescapsular polysaccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V,14, 18C, 19F and 23F (preferably conjugated). In a preferred embodimentof the invention at least 13 polysaccharide antigens (preferablyconjugated) are included, although further polysaccharide antigens, forexample 23 valent (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V,10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), arealso contemplated by the invention.

For elderly vaccination (for instance for the prevention of pneumonia)it is advantageous to include serotypes 8 and 12F (and most preferably15 and 22 as well) to the 11 valent antigenic composition describedabove to form a 15 valent vaccine, whereas for infants or toddlers(where otitis media is of more concern) serotypes 6A and 19A areadvantageously included to form a 13 valent vaccine.

For the prevention/amelioration of pneumonia in the elderly (+55 years)population and Otitis media in Infants (up to 18 months) and toddlers(typically 18 months to 5 years), it is a preferred embodiment of theinvention to combine a multivalent Streptococcus pneumoniapolysaccharide as herein described with a Streptococcus pneumoniaeprotein or immunologically functional equivalent thereof.

Pneumococcal Proteins of the Invention

For the purposes of this invention, “immunologically functionalequivalent” is defined as a peptide of protein comprising at least oneprotective epitope from the proteins of the invention. Such epitopes arecharacteristically surface-exposed, highly conserved, and can elicit anbactericidal antibody response in a host or prevent toxic effects.Preferably, the functional equivalent has at least 15 and preferably 30or more contiguous amino acids from the protein of the invention. Mostpreferably, fragments, deletions of the protein, such as transmembranedeletion variants thereof (ie the use of the extracellular domain of theproteins), fusions, chemically or genetically detoxified derivatives andthe like can be used with the proviso that they are capable of raisingsubstantially the same immune response as the native protein.

Preferred proteins of the invention are those pneumococcal proteinswhich are exposed on the outer surface of the pneumococcus (capable ofbeing recognised by a host's immune system during at least part of thelife cycle of the pneumococcus), or are proteins which are secreted orreleased by the pneumococcus. Most preferably, the protein is a toxin,adhesin, 2-component signal tranducer, or lipoprotein of Streptococcuspneumoniae, or immunologically functional equivalents thereof.

Particularly preferred proteins to be included in such a combinationvaccine, include but are not limited to: pneumolysin (preferablydetoxified by chemical treatment or mutation) [Mitchell et al. NucleicAcids Res. 1990 Jul. 11; 18(13): 4010 “Comparison of pneumolysin genesand proteins from Streptococcus pneumoniae types 1 and 2.”, Mitchell etal. Biochim Biophys Acta 1989 Jan. 23; 1007(1): 67-72 “Expression of thepneumolysin gene in Escherichia coli: rapid purification and biologicalproperties.”, WO 96/05859 (A. Cyanamid), WO 90/06951 (Paton et al), WO99/03884 (NAVA)]; PspA and transmembrane deletion variants thereof (U.S.Pat. No. 5,804,193—Briles et al.); PspC and transmembrane deletionvariants thereof (WO 97/09994—Briles et al); PsaA and transmembranedeletion variants thereof (Berry & Paton, Infect Immun 1996 December;64(12):5255-62 “Sequence heterogeneity of PsaA, a 37-kilodalton putativeadhesin essential for virulence of Streptococcus pneumoniae”);pneumococcal choline binding proteins and transmembrane deletionvariants thereof; CbpA and transmembrane deletion variants thereof (WO97/41151; WO 99/51266); Glyceraldehyde-3-phosphate—dehydrogenase(Infect. Immun. 1996 64:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beatoet al. FEMS Microbiol Lett 1998, 164:207-14); M like protein, SB patentapplication No. EP 0837130; and adhesin 18627, SB Patent application No.EP 0834568.

The proteins used in the present invention are preferably selected fromthe group pneumolysin, PsaA, PspA, PspC, CbpA or a combination of two ormore such proteins. The present invention also encompassesimmunologically functional equivalents of such proteins (as definedabove).

Within the composition, the protein can help to induce a T-cell mediatedresponse against pneumococcal disease—particularly required forprotection against pneumonia—which cooperates with the humoral branch ofthe immune system to inhibit invasion by pneumococci, and to stimulateopsonophagocytosis.

Further advantages of including the protein antigen is presentation offurther antigens for the opsonophagocytosis process, and the inhibitionof bacterial adhesion (if an adhesin is used) or the neutralisation oftoxin (if a toxin is used).

Accordingly in an embodiment of the invention there is provided aStreptococcus pneumoniae vaccine comprising a pneumococcuspolysaccharide conjugate vaccine comprising polysaccharide antigensderived from at least four serotypes, preferably at least sevenserotypes, more preferably at least eleven serotypes, and at least one,but preferably two, Streptococcus pneumoniae proteins. Preferably one ofthe proteins is Pneumolysin or PsaA or PspA or CbpA (most preferablydetoxified pneumolysin). A preferred combination contains at leastpneumolysin or a derivative thereof and PspA.

As mentioned above, a problem associated with the polysaccharideapproach to vaccination, is the fact that polysaccharides per se arepoor immunogens. To overcome this, polysaccharides may be conjugated toprotein carriers, which provide bystander T-cell help. It is preferred,therefore, that the polysaccharides utilised in the invention are linkedto such a protein carrier. Examples of such carriers which are currentlycommonly used for the production of polysaccharide immunogens includethe Diphtheria and Tetanus toxoids (DT, DT CRM197 and TT respectively),Keyhole Limpet Haemocyanin (KLH), OMPC from N. meningitidis, and thepurified protein derivative of Tuberculin (PPD).

A number of problems are, however, associated with each of thesecommonly used carriers (see section “Problems Associated withCommonly-Used Carriers” above).

The present invention provides in a preferred embodiment a new carrierfor use in the preparation of polysaccharide-based immunogen constructs,that does not suffer from these disadvantages. The preferred carrier forthe pneumococcal polysaccharide based immunogenic compositions (orvaccines) is protein D from Haemophilus influenzae (EP 594610-B), orfragments thereof. Fragments suitable for use include fragmentsencompassing T-helper epitopes. In particular a protein D fragment willpreferably contain the N-terminal ⅓ of the protein.

A further preferred carrier for the pneumococcal polysaccharide is thepneumococcal protein itself (as defined above in section “PneumococcalProteins of the invention”).

The vaccines of the present invention are preferably adjuvanted.Suitable adjuvants include an aluminium salt such as aluminium hydroxidegel (alum) or aluminium phosphate, but may also be a salt of calcium,iron or zinc, or may be an insoluble suspension of acylated tyrosine, oracylated sugars, cationically or anionically derivatisedpolysaccharides, or polyphosphazenes.

It is preferred that the adjuvant be selected to be a preferentialinducer of a TH1 type of response to aid the cell mediated branch of theimmune response.

TH1 Adjuvants of the Invention

High levels of Th1-type cytokines tend to favour the induction of cellmediated immune responses to a given antigen, whilst high levels ofTh2-type cytokines tend to favour the induction of humoral immuneresponses to the antigen.

It is important to remember that the distinction of Th1 and Th2-typeimmune response is not absolute. In reality an individual will supportan immune response which is described as being predominantly Th1 orpredominantly Th2. However, it is often convenient to consider thefamilies of cytokines in terms of that described in murine CD4+ve T cellclones by Mosmann and Coffman (Mosmann, T. R. and Coffman, R. L. (1989)TH1 and TH2 cells: different patterns of lymphokine secretion lead todifferent functional properties. Annual Review of Immunology, 7,p145-173). Traditionally, Th1-type responses are associated with theproduction of the INF-γ and IL-2 cytokines by T-lymphocytes. Othercytokines often directly associated with the induction of Th1-typeimmune responses are not produced by T-cells, such as IL-12. Incontrast, Th2-type responses are associated with the secretion of 11-4,IL-5, IL-6, IL-10. Suitable adjuvant systems which promote apredominantly Th1 response include, Monophosphoryl lipid A or aderivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A,and a combination of monophosphoryl lipid A, preferably 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) together with an aluminium salt.

An enhanced system involves the combination of a monophosphoryl lipid Aand a saponin derivative, particularly the combination of QS21 and3D-MPL as disclosed in WO 94/00153, or a less reactogenic compositionwhere the QS21 is quenched with cholesterol as disclosed in WO 96/33739.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210, andis a preferred formulation.

Preferably the vaccine additionally comprises a saponin, more preferablyQS21. The formulation may also comprises an oil in water emulsion andtocopherol (WO 95/17210).

The present invention also provides a method for producing a vaccineformulation comprising mixing a protein of the present inventiontogether with a pharmaceutically acceptable excipient, such as 3D-MPL.

Unmethylated CpG containing oligonucleotides (WO 96/02555) are alsopreferential inducers of a TH1 response and are suitable for use in thepresent invention.

Particularly preferred compositions of the invention comprise one ormore conjugated pneumococcal polysaccharides, one or more pneumococcalproteins and a Th1 adjuvant. The induction of a cell mediated responseby way of a pneumococcal protein (as described above) and thecooperation between both arms of the immuen system may be aided usingsuch a Th-1 adjuvant, resulting in a particularly effective vaccineagainst pneumococcal disease in general, and, importantly, againstpneumococcal pneumonia in the elderly.

In a further aspect of the present invention there is provided animmunogen or vaccine as herein described for use in medicine.

In a still further aspect of the invention, a composition is providedcomprising a pneumococcal polysaccharide conjugate and a Th1 adjuvant(preferably 3D-MPL) which is capable of seroconverting or inducing ahumoral antibody response against the polysaccharide antigen within apopulation of non-responders.

10-30% of the population are known to be non-responders topolysaccharide immunization (do not respond to more than 50% ofserotypes in a vaccine) (Konradsen et al., Scand. J. Immun 40:251(1994); Rodriguez et al., JID, 173:1347 (1996)). This can also be thecase for conjugated vaccines (Musher et al. Clin. Inf. Dis. 27:1487(1998)). This can be particularly serious for high risk areas of thepopulation (infants, toddlers and the elderly).

The present inventors have found that a combination of a conjugatedpneumococcal polysaccharide (which is prone to low response in aparticular population) with a Th1 adjuvant (see “Th1 adjuvants of theinvention” above) can surprisingly overcome this non-responsiveness.Preferably 3D-MPL should be used, and most preferably 3D-MPL devoid ofaluminium-based adjuvant (which provides a better response still). Thepresent invention thus provides such compositions, and further providesa method of treating non-responders to pneumococcal polysaccharides byadministering such compositions, and still further provides a use of aTh1 adjuvant in the manufacture of a medicament comprising conjugatedpneumococcal polysaccharide antigens, in the treatment against (orprotection from) pneumococcal disease in individuals which arenon-responsive to the polysaccharide antigen.

In one embodiment there is a method of preventing or amelioratingpneumonia in an elderly human comprising administering a safe andeffective amount of a vaccine, as described herein, comprising aStreptoccocus pneumoniae polysaccharide antigen and either a Th1adjuvant, or a pneumococcal protein (and preferably both), to saidelderly patient.

In a further embodiment there is provided a method of preventing orameliorating otitis media in Infants or toddlers, comprisingadministering a safe and effective amount of a vaccine comprising aStreptococcus pneumoniae polysaccharide antigen and either aStreptococcus pneumoniae protein antigen or a Th1 adjuvant (andpreferably both), to said Infant or toddler.

Preferably in the methods of the invention as described above thepolysaccharide antigen is present as a polysaccharide protein conjugate.

Vaccine Preparations of the Invention

The vaccine preparations of the present invention may be used to protector treat a mammal susceptible to infection, by means of administeringsaid vaccine via systemic or mucosal route. These administrations mayinclude injection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. Intranasaladministration of vaccines for the treatment of pneumonia or otitismedia is preferred (as nasopharyngeal carriage of pneumococci can bemore effectively prevented, thus attenuating infection at its earlieststage).

The amount of conjugate antigen in each vaccine dose is selected as anamount which induces an immunoprotective response without significant,adverse side effects in typical vaccines. Such amount will varydepending upon which specific immunogen is employed and how it ispresented. Generally, it is expected that each dose will comprise0.1-100 μg of polysaccharide, preferably 0.1-50 μg, preferably 0.1-10μg, of which 1 to 5 μg is the most preferable range.

The content of protein antigens in the vaccine will typically be in therange 1-100 μg, preferably 5-50 μg, most typically in the range 5-25 μg.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. Following an initial vaccination, subjectsmay receive one or several booster immunisations adequately spaced.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

B) Selected Pneumococcal Polysaccharide Conjugate+3D-MPL Compositions

For the purposes of this invention, the term “pneumococcalpolysaccharide conjugates of the invention” describes those conjugatesof Streptococcus pneumoniae capsular polysaccharides which are moreimmunogenic in compositions comprising 3D-MPL in comparison withcompositions comprising 3D-MPL in conjunction with an aluminium-basedadjuvant (for example, conjugates of serotype 4; serotype 6B; serotype18C; serotype 19F; or serotype 23F).

For the purposes of this invention, the term “substantially devoid ofaluminium-based adjuvants” describes a composition which does notcontain sufficient aluminium-based adjuvant (for example aluminiumhydroxide, and, particularly, aluminium phosphate) to cause any decreasein the immunogenicity of a pneumococcal polysaccharide conjugate of theinvention in comparison to an equivalent composition comprising 3D-MPLwith no added aluminium-based adjuvant. Preferably the antigeniccomposition should contain adjuvant that consists essentially of 3D-MPL.Quantities of aluminium-based adjuvant added per dose should preferablybe less than 50 μg, more preferably less than 30 μg, still morepreferably less than 10 μg, and most preferably there is noaluminium-based adjuvant added to the antigenic compositions of theinvention.

For the purposes of this invention, the determination of whether apneumococcal polysaccharide conjugate is significantly more immunogenicin compositions comprising 3D-MPL in comparison with compositionscomprising 3D-MPL in conjunction with an aluminium-based adjuvant, thisshould be established as described in Example 2. As an indication ofwhether a composition is significantly more immunogenic when comprising3D-MPL alone, the ratio of GMC IgG concentration (as determined inExample 2) between compositions comprising 3D-MPL alone versus anequivalent composition comprising 3D-MPL in conjunction with aluminiumphosphate adjuvant should be more than 2, preferably more than 5, morepreferably more than 7, still more preferably more than 9, and mostpreferably more than 14.

Amongst the problems associated with the polysaccharide approach tovaccination, is the fact that polysaccharides per se are poorimmunogens. Strategies, which have been designed to overcome this lackof immunogenicity, include the linking (conjugating) of thepolysaccharide to large protein carriers, which provide bystander T-cellhelp. It is preferred that the pneumococcal polysaccharides of theinvention are linked to a protein carrier which provides bystanderT-cell help. Examples of such carriers which may be used include theDiphtheria, Diphtheria mutant, and Tetanus toxoids (DT, CRM197 and TTrespectively), Keyhole Limpet Haemocyanin (KLH), the purified proteinderivative of Tuberculin (PPD), and OMPC of Neisseria meningitidis.

Most preferably, protein D from Haemophilus influenzae (EP 0 594 610-B),or fragments thereof (see section C), is used as the immunogenic proteincarrier for the pneumococcal polysaccharides of the invention.

In one embodiment the antigenic composition of the invention comprisespneumococcal polysaccharide serotype (PS) 4 conjugated to an immunogenicprotein and formulated with 3D-MPL adjuvant, where the composition issubstantially devoid of aluminium-based adjuvant. In furtherembodiments, the antigenic composition comprises PS 6B, 18C, 19F, or23F, respectively, conjugated to an immunogenic protein and formulatedwith 3D-MPL adjuvant, where the composition is substantially devoid ofaluminium-based adjuvant.

In a still further embodiment of the invention, a combination antigeniccomposition is provided comprising two or more pneumococcalpolysaccharide conjugates from the group PS 4, PS 6B, PS18C, PS19F, andPS 23F formulated with 3D-MPL adjuvant, where the composition issubstantially devoid of aluminium-based adjuvant.

The immunogenicity of pneumococcal polysaccharide conjugates of theinvention is not significantly effected by combination with otherpneumococcal polysaccharide conjugates (Example 3). Accordingly, apreferred aspect of the invention provides a combination antigeniccomposition comprising one or more pneumococcal polysaccharideconjugates of the invention in combination with one or more furtherpneumococcal polysaccharide conjugates, where the composition isformulated with 3D-MPL adjuvant, but is substantially devoid ofaluminium-based adjuvant.

In further preferred embodiments of the invention, combination antigeniccompositions are provided which contain at least one and preferably 2,3, 4 or all 5 of the PS 4, 6B, 18C, 19F, or 23F pneumococcalpolysaccharide conjugates, and in addition any combination of otherpneumococcal polysaccharide conjugates, which are formulated with 3D-MPLadjuvant but substantially devoid of aluminium-based adjuvant.

Typically the Streptococcus pneumoniae combination antigenic compositionof the present invention will comprise polysaccharide conjugateantigens, wherein the polysaccharides are derived from at least four,seven, eleven, thirteen, fifteen or twenty-three serotypes (see“Streptococcus pneumoniae Polysaccharide Antigens of the Invention”above for preferred combinations of serotypes depending on the diseaseto be treated).

The antigenic compositions of the invention are preferably used asvaccine compositions to prevent (or treat) pneumococcal infections,particularly in the elderly and infants and toddlers.

Further embodiments of the present invention include: the provision ofthe above antigenic compositions for use in medicine; a method ofinducing an immune response to a Streptococcus pneumoniae capsularpolysaccharide conjugate, comprising the steps of administering a safeand effective amount of one of the above antigenic compositions to apatient; and the use of one of the above antigenic compositions in themanufacture of a medicament for the prevention (or treatment) ofpneumococcal disease.

For the prevention/amelioration of pneumonia in the elderly (+55 years)population and Otitis media in Infants (up to 18 months) and toddlers(typically 18 months to 5 years), it is a further preferred embodimentof the invention to combine a multivalent Streptococcus pneumoniapolysaccharide conjugate formulated as herein described with aStreptococcus pneumoniae protein or immunologically functionalequivalent thereof. See above section “Pneumococcal Proteins of theinvention” for preferred proteins/protein combinations.

Preferably the antigenic compositions (and vaccines) hereinbeforedescribed are lyophilised up until they are about to be used, at whichpoint they are extemporaneously reconstituted with diluent. Morepreferably they are lyophilsed in the presence of 3D-MPL, and areextemporaneously reconstituted with saline solution.

Lyophilising the compositions results in a more stable composition (forinstance it prevents the breakdown of the polysaccharide antigens). Theprocess is also surprisingly responsible for a higher antibody titrestill against the pneumococcal polysaccharides. This has been shown tobe particularly significant for PS 6B conjugates. Another aspect of theinvention is thus a lyophilised antigenic composition comprising a PS 6Bconjugate adjuvanted with 3D-MPL and substantially devoid ofaluminium-based adjuvants.

For preparation of the vaccines, see above “Vaccine Preparations of theInvention” section.

C) Bacterial Polysaccharide—Protein D Conjugates

The trend towards combination vaccines has the advantage of reducingdiscomfort to the recipient, facilitating scheduling, and ensuringcompletion of regiment; but there is also the concomitant risk ofreducing the vaccine's efficacy (see above for discussion on epitopesuppression through overuse of carrier proteins). It would be,therefore, advantageous to make vaccine combinations which meet theneeds of a population, and which, in addition, do not exhibitimmunogenic interference between their components. These advantages maybe realised by the immunogenic compositions (or vaccines) of theinvention, which are of particular benefit for administration ofcombination vaccines to high risk groups such infants, toddlers or theelderly.

The present invention provides a protein D from Haemophilus influenzae,or fragments thereof, as a carrier for polysaccharide based immunogeniccomposition, including vaccines. Fragments suitable for use includefragments encompassing T-helper epitopes. In particular protein Dfragment will, preferably contain the N-terminal ⅓ of the protein.

Protein D is an IgD-binding protein from Haemophilus influenzae (EP 0594 610 B1) and is a potential immunogen.

Polysaccharides to be conjugated to Protein D contemplated by thepresent invention include, but are not limited to the Vi polysaccharideantigen against Salmonella typhi, meningococcal polysaccharides(including type A, C, W135 and Y, and the polysaccharide and modifiedpolysaccharides of group B meningococcus), polysaccharides' fromStaphylococcus aureus, polysaccharides from Streptococcus agalactae,polysaccharides from Streptococcus pneumoniae, polysaccharides fromMycobacterium e.g. Mycobacterium tuberculosis (such asmannophosphoinisitides trehaloses, mycolic acid, mannose cappedarabinomannans, the capsule therefrom and arabinogalactans),polysaccharide from Cryptococcus neoformans, the lipopolysaccharides ofnon-typeable Haemophilus influenzae, the capsular polysaccharide fromHaemophilus influenzae b, the lipopolysaccharides of Moraxellacatharralis, the lipopolysaccharides of Shigella sonnei, thelipopeptidophosphoglycan (LPPG) of Trypanosoma cruzi, the cancerassociated gangliosides GD3, GD2, the tumor associated mucins,especially the T-F antigen, and the sialyl T-F antigen, and the HIVassociated polysaccharide that is structurally related to the T-Fantigen.

The polysaccharide may be linked to the carrier protein by any knownmethod (for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor etal., U.S. Pat. No. 4,474,757). Preferably, CDAP conjugation is carriedout (WO 95/08348).

In CDAP, the cyanylating reagent 1-cyano-dimethylaminopyridiniumtetrafluoroborate (CDAP) is preferably used for the synthesis ofpolysaccharide-protein conjugates. The cyanilation reaction can beperformed under relatively mild conditions, which avoids hydrolysis ofthe alkaline sensitive polysaccharides. This synthesis allows directcoupling to a carrier protein.

The polysaccharide is solubilized in water or a saline solution. CDAP isdissolved in acetonitrile and added immediately to the polysaccharidesolution. The CDAP reacts with the hydroxyl groups of the polysaccharideto form a cyanate ester. After the activation step, the carrier proteinis added. Amino groups of lysine react with the activated polysaccharideto form an isourea covalent link.

After the coupling reaction, a large excess of glycine is then added toquench residual activated functions. The product is then passed througha gel permeation to remove unreacted carrier protein and residualreagents. Accordingly the invention provides a method of producingpolysaccharide protein D conjugates comprising the steps of activatingthe polysaccharide and linking the polysaccharide to the protein D.

In a preferred embodiment of the invention there is provided animmunogenic composition (or vaccine) formulation for the prevention ofStreptococcus pneumoniae infections.

The mechanisms by which pneumococci spread to the lung, thecerebrospinal fluid and the blood is poorly understood. Growth ofbacteria reaching normal lung alveoli is inhibited by their relativedryness and by the phagocytic activity of alveolar macrophages. Anyanatomic or physiological changes of these co-ordinated defences tend toaugment the susceptibility of the lungs to infection. The cell-wall ofStreptococcus pneumoniae has an important role in generating aninflammatory response in the alveoli of the lung (Gillespie et al.(1997), I&I 65: 3936).

Typically the Streptococcus pneumoniae vaccine of the present inventionwill comprise protein D polysaccharide conjugates, wherein thepolysaccharide is derived from at least four, seven, eleven, thirteen,fifteen or 23 serotypes. See above “Streptococcus pneumoniaePolysaccharide Antigens of the Invention” for preferred combinations ofserotypes depending on the disease to be treated.

In a further embodiment of the invention there is provided a Neisseriameningitidis vaccine; in particular from serotypes A, B, C W-135 and Y.Neisseria meningitidis is one of the most important causes of bacterialmeningitis. The carbohydrate capsule of these organisms can act as avirulence determinant and a target for protective antibody.Carbohydrates are nevertheless well known to be poor immunogens in youngchildren. The present invention provides a particularly suitable proteincarrier for these polysaccharides, protein D, which provides T-cellepitopes that can activate a T-cell response to aid polysaccharideantigen specific B-cell proliferation and maturation, as well as theinduction of an immunological memory.

In an alternative embodiment of the invention there is provided acapsular polysaccharide of Haemophilus influenzae b (PRP)—protein Dconjugate.

The present invention also contemplates combination vaccines whichprovide protection against a range of different pathogens. A protein Dcarrier is surprisingly useful as a carrier in combination vaccineswhere multiple polysaccharide antigens are conjugated. As mentionedabove, epitope suppression is likely to occur if the same carrier isused for each polysaccharide. WO 98/51339 presented compositions to tryto minimise this interference by conjugating a proportion of thepolysaccharides in the composition onto DT and the rest onto TT.

Surprisingly, the present inventors have found protein D is particularlysuitable for minimising such epitopic suppression effects in combinationvaccines. One or more polysaccharides in a combination may beadvantageously conjugated onto protein D, and preferably all antigensare conjugated onto protein D within such combination vaccines.

A preferred combination includes a vaccine that affords protectionagainst Neisseria meningitidis C and Y (and preferably A) infectionwherein the polysaccharide antigen from one or more of serotypes Y and C(and most preferably A) are linked to protein D.

Haemophilus influenzae polysaccharide based vaccine (PRP conjugated withpreferably TT, DT or CRM197, or most preferably with protein D) may beformulated with the above combination vaccines.

Many Paediatric vaccines are now given as a combination vaccine so as toreduce the number of injections a child has to receive. Thus forPaediatric vaccines other antigens may be formulated with the vaccinesof the invention. For example the vaccines of the invention can beformulated with, or administered separately, but at the same time withthe well known ‘trivalent’ combination vaccine comprising Diphtheriatoxoid (DT), tetanus toxoid (CT), and pertussis components [typicallydetoxified Pertussis toxoid (PT) and filamentous haemagglutinin (FHA)with optional pertactin (PRN) and/or agglutinin 1+2], for example themarketed vaccine INFANRIX-DTPa™ (SmithKlineBeecham Biologicals) whichcontains DT, TT, PT, FHA and PRN antigens, or with a whole cellpertussis component for example as marketed by SmithKlineBeechamBiologicals s.a., as TRITANRIX™. The combined vaccine may also compriseother antigen, such as Hepatitis B surface antigen (HBsAg), Polio virusantigens (for instance inactivated trivalent polio virus—IPV), Moraxellacatarrhalis outer membrane proteins, non-typeable Haemophilus influenzaeproteins, N. meningitidis B outer membrane proteins.

Examples of preferred Moraxella catarrhalis protein antigens which canbe included in a combination vaccine (especially for the prevention ofotitis media) are: OMP106 [WO 97/41731 (Antex) & WO 96/34960 (PMC)];OMP21; LbpA & LbpB [WO 98/55606 (PMC)]; TbpA & TbpB [WO 97/13785 & WO97/32980 (PMC)]; CopB [Helminen M E, et al. (1993) Infect. Immun.61:2003-2010]; UspA1/2 [WO 93/03761 (University of Texas)]; and OmpCD.Examples of non-typeable Haemophilus influenzae antigens which can beincluded in a combination vaccine (especially for the prevention ofotitis media) include: Fimbrin protein [(U.S. Pat. No. 5,766,608—OhioState Research Foundation)] and fusions comprising peptides therefrom[eg LB1(f) peptide fusions; U.S. Pat. No. 5,843,464 (OSU) or WO99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673 (StateUniversity of New York)]; TbpA and TbpB; Hia; Hmw1,2; Hap; and D15.

Preferred Peadiatric vaccines contemplated by the present invention are:

-   a) N. meningitidis C polysaccharide conjugate and Haemophilus    influenzae b polysaccharide conjugate, optionally with N.    meningitidis A and/or Y polysaccharide conjugate, provided that at    least one polysaccharide antigen, and preferably all are conjugated    to protein D.-   b) Vaccine a) with, DT, TT, pertussis components (preferable PT, FHA    and PRN), Hepatitis B surface antigen and IPV (inactivated trivalent    poliovirus vaccine).-   c) Streptococcus pneumoniae polysaccharide antigens conjugated to    protein D.-   d) Vaccine c) with one or more antigens from Moraxella catarrhalis    and/or non-typeable Haemophilus influenzae.

All the above combination vaccines, can benefit from the inclusion ofprotein D as a carrier. Clearly, the more carriers that are involved ina combination vaccine (for instance to overcome epitope suppression),the more expensive and complex the final vaccine. Having all, or themajority, of the polysaccharide antigens of a combination vaccineconjugated to protein D thus provides a considerable advantage

For the prevention of pneumonia in the elderly (+55 years) populationand Otitis media in Infants or toddlers, it is a preferred embodiment ofthe invention to combine a multivalent streptococcus pneumoniapolysaccharide—protein D antigens as herein described with aStreptococcus pneumoniae protein or immunologically functionalequivalent thereof. See above section “Pneumococcal Proteins of theinvention” for preferred proteins/protein combinations that can beincluded in such a combination.

Accordingly the present invention provides an immunogenic compositioncomprising a Streptococcus pneumoniae polysaccharide—protein D conjugateand a Streptococcus pneumoniae protein antigen.

The polysaccharide—protein D conjugate antigens of the present inventionare preferably adjuvanted in the vaccine formulation of the invention.Suitable adjuvants include an aluminium salt such as aluminum hydroxidegel (alum) or aluminium phosphate, but may also be a salt of calcium,iron or zinc, or may be an insoluble suspension of acylated tyrosine, oracylated sugars, cationically or anionically derivatisedpolysaccharides, or polyphosphazenes.

For elderly vaccines it is preferred that the adjuvant be selected to bea preferential inducer of a TH1 type of response.

For particular Th1 adjuvants see “Th1 adjuvants of the invention” above.

In a further aspect of the present invention there is provided animmunogen or vaccine as herein described for use in medicine.

For vaccine preparation/administration of the conjugate, see “VaccinePreparation of the Invention” above.

Protein D is also advantageously used in a vaccine against otitis media,as it is in itself an immunogen capable of producing B-cell mediatedprotection against non-typeable H. influenzae (ntHi). ntHi may invadehost cells, and evade the B-cell mediated effects induced by the proteinantigen. The present inventors have surprisingly found a way ofincreasing the effectiveness of protein D (either by itself or as acarrier for a polysaccharide) as an antigen for an otitis media vaccine.This is done by adjuvanting the protein D such that a strong Th1response is induced in the subject such that the cell mediated arm ofthe immune system is optimised against protein D. This is surprisinglyachieved using a lyophilised composition comprising protein D and a Th1adjuvant (preferably 3D-MPL) which is reconstituted shortly beforeadministration. The invention thus also provides such compositions, aprocess for making such compositions (by lyophilising a mixturecomprising protein D and a Th1 adjuvant), and a use of such acomposition in the treatment of otitis media.

In a broader sense, the inventors envisage that lyophilising animmunogen in the presence of a Th1 adjuvant (see “Th1 adjuvants of theinvention”), preferably 3D-MPL, will generally augment the Th1 immuneresponse against the immunogen. The present invention is thereforeapplicable to any immunogen to which a stronger Th1 immune response isrequired. Such immunogens comprise bacterial, viral and tumour proteinantigens, as well as self proteins and peptides.

EXAMPLES

The examples illustrate, but do not limit the invention.

Example 1 S. pneumoniae Capsular Polysaccharide

The 11-valent candidate vaccine includes the capsular polysaccharidesserotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F which were madeessentially as described in EP 72513. Each polysaccharide is activatedand derivatised using CDAP chemistry (WO 95/08348) and conjugated to theprotein carrier. All the polysaccharides are conjugated in their nativeform, except for the serotype 3 (which was size-reduced to decrease itsviscosity).

Protein Carrier:

The protein carrier selected is the recombinant protein D (PD) from Nontypeable Haemophilus influenzae, expressed in E. coli.

Expression of Protein D

Haemophilus influenzae Protein D

Genetic Construction for Protein D Expression Starting Materials TheProtein D Encoding DNA

Protein D is highly conserved among H. influenzae of all serotypes andnon-typeable strains. The vector pHIC348 containing the DNA sequenceencoding the entire protein D gene has been obtained from Dr. A.Forsgren, Department of Medical Microbiology, University of Lund, MalmöGeneral Hospital, Malmö, Sweden. The DNA sequence of protein D has beenpublished by Janson et al. (1991) Infect. Immun. 59: 119-125.

The Expression Vector pMG1

The expression vector pMG1 is a derivative of pBR322 (Gross et al.,1985) in which bacteriophage λ derived control elements fortranscription and translation of foreign inserted genes were introduced(Shatzman et al., 1983). In addition, the Ampicillin resistance gene wasexchanged with the Kanamycin resistance gene.

The E. coli Strain AR58

The E. coli strain AR58 was generated by transduction of N99 with a P1phage stock previously grown on an SA500 derivative (galE::TNIO,lambdaKil⁻ cI857 ΔH1). N99 and SA500 are E. coli K12 strains derivedfrom Dr. Martin Rosenberg's laboratory at the National Institute ofHealth.

The Expression Vector pMG1

For the production of protein D, the DNA encoding the protein has beencloned into the expression vector pMG1. This plasmid utilises signalsfrom lambdaphage DNA to drive the transcription and translation ofinserted foreign genes. The vector contains the promoter PL, operator OLand two utilisation sites (NutL and NutR) to relieve transcriptionalpolarity effects when N protein is provided (Gross et al., 1985).Vectors containing the PL promoter, are introduced into an E. colilysogenic host to stabilise the plasmid DNA. Lysogenic host strainscontain replication-defective lambdaphage DNA integrated into the genome(Shatzman et al., 1983). The chromosomal lambdaphage DNA directs thesynthesis of the cI repressor protein which binds to the OL repressor ofthe vector and prevents binding of RNA polymerase to the PL promoter andthereby transcription of the inserted gene. The cI gene of theexpression strain AR58 contains a temperature sensitive mutant so thatPL directed transcription can be regulated by temperature shift, i.e. anincrease in culture temperature inactivates the repressor and synthesisof the foreign protein is initiated. This expression system allowscontrolled synthesis of foreign proteins especially of those that may betoxic to the cell (Shimataka & Rosenberg, 1981).

The E. coli Strain AR58

The AR58 lysogenic E. coli strain used for the production of the proteinD carrier is a derivative of the standard NIH E. coli K12 strain N99 (F⁻su⁻ galK2, lacZ⁻ thr⁻). It contains a defective lysogenic lambdaphage(galE::TNIO, lambdaKil⁻ cI857 ΔH1). The Kil⁻ phenotype prevents the shutoff of host macromolecular synthesis. The cI857 mutation confers atemperature sensitive lesion to the cI repressor. The ΔH1 deletionremoves the lambdaphage right operon and the hosts bio, uvr3, and chlAloci. The AR58 strain was generated by transduction of N99 with a P1phage stock previously grown on an SA500 derivative (galE::TNIO,lambdaKil⁻ cI857 ΔH1). The introduction of the defective lysogen intoN99 was selected with tetracycline by virtue of the presence of a TN10transposon coding for tetracyclin resistance in the adjacent galE gene.

Construction of Vector pMGMDPPrD

The pMG 1 vector which contains the gene encoding the non-structural S1protein of Influenzae virus (pMGNSI) was used to construct pMGMDPPrD.The protein D gene was amplified by PCR from the pHIC348 vector (Jansonet al. 1991) with PCR primers containing NcoI and XbaI restriction sitesat the 5′ and 3′ ends, respectively. The NcoI/XbaI fragment was thenintroduced into pMGNS1 between NcoI and XbaI thus creating a fusionprotein containing the N-terminal 81 amino acids of the NS1 proteinfollowed by the PD protein. This vector was labeled pMGNS1PrD.

Based on the construct described above the final construct for protein Dexpression was generated. A BamHI/BamHI fragment was removed frompMGNS1PrD. This DNA hydrolysis removes the NS1 coding region, except forthe first three N-terminal residues. Upon religation of the vector agene encoding a fusion protein with the following N-terminal amino acidsequence has been generated:

-----MDP SSHSSNMANT----- (SEQ ID NO: 1) NS1               Protein D

The protein D does not contain a leader peptide or the N-terminalcysteine to which lipid chains are normally attached. The protein istherefore neither excreted into the periplasm nor lipidated and remainsin the cytoplasm in a soluble form.

The final construct pMG-MDPPrD was introduced into the AR58 host strainby heat shock at 37° C. Plasmid containing bacteria were selected in thepresence of Kanamycin. Presence of the protein D encoding DNA insert wasdemonstrated by digestion of isolated plasmid DNA with selectedendonucleases. The recombinant E. coli strain is referred to as ECD4.

Expression of protein D is under the control of the lambda P_(L)promoter/O_(L) Operator. The host strain AR58 contains atemperature-sensitive cI gene in the genome which blocks expression fromlambda P_(L) at low temperature by binding to O_(L). Once thetemperature is elevated c1 is released from O_(L) and protein D isexpressed. At the end of the fermentation the cells are concentrated andfrozen.

The extraction from harvested cells and the purification of protein Dwas performed as follows. The frozen cell culture pellet is thawed andresuspended in a cell disruption solution (Citrate buffer pH 6.0) to afinal OD₆₅₀=60. The suspension is passed twice through a high pressurehomogenizer at P=1000 bar. The cell culture homogenate is clarified bycentrifugation and cell debris are removed by filtration. In the firstpurification step the filtered lysate is applied to a cation exchangechromatography column (SP SEPHAROSE® Fast Flow). PD binds to the gelmatrix by ionic interaction and is eluted by a step increase of theionic strength of the elution buffer.

In a second purification step impurities are retained on an anionicexchange matrix (Q SEPHAROSE® Fast Flow). PD does not bind onto the geland can be collected in the flow through.

In both column chromatography steps fraction collection is monitored byOD. The flow through of the anionic exchange column chromatographycontaining the purified protein D is concentrated by ultrafiltration.

The protein D containing ultrafiltration retentate is finally passedthrough a 0.2 μm membrane.

Chemistry: Activation and Coupling Chemistry:

The activation and coupling conditions are specific for eachpolysaccharide. These are given in Table 1. Native polysaccharide(except for PS3) was dissolved in NaCl 2M or in water for injection. Theoptimal polysaccharide concentration was evaluated for all theserotypes.

From a 100 mg/ml stock solution in acetonitrile, CDAP (CDAP/PS ratio0.75 mg/mg PS) was added to the polysaccharide solution. 1.5 minutelater, 0.2M triethylamine was added to obtain the specific activationpH. The activation of the polysaccharide was performed at this pH during2 minutes at 25° C. Protein D (the quantity depends on the initial PS/PDratio) was added to the activated polysaccharide and the couplingreaction was performed at the specific pH for 1 hour. The reaction wasthen quenched with glycine for 30 minutes at 25° C. and overnight at 4°C.

The conjugates were purified by gel filtration using a SEPHACRYL® 500HRgel filtration column equilibrated with 0.2M NaCl.

The carbohydrate and protein content of the eluted fractions wasdetermined. The conjugates were pooled and sterile filtered on a 0.22 μmsterilizing membrane. The PS/Protein ratios in the conjugatepreparations were determined.

Characterisation:

Each conjugate was characterised and met the specifications described inTable 2. The polysaccharide content (μg/ml) was measured by theResorcinol test and the protein content (μg/ml) by the Lowry test. Thefinal PS/PD ratio (w/w) is determined by the ratio of theconcentrations.

Residual DMAP Content (ng/μg PS):

The activation of the polysaccharide with CDAP introduces a cyanategroup in the polysaccharide and DMAP (4-dimethylamino-pyridin) isliberated. The residual DMAP content was determined by a specific assaydeveloped at SB.

Free Polysaccharide Content (%):

The free polysaccharide content of conjugates kept at 4° C. or stored 7days at 37° C. was determined on the supernatant obtained afterincubation with α-PD antibodies and saturated ammonium sulfate, followedby a centrifugation.

An α-PS/α-PS ELISA was used for the quantification of freepolysaccharide in the supernatant. The absence of conjugate was alsocontrolled by an α-PD/α-PS ELISA. Reducing the quantity of freepolysaccharide results in an improved conjugate vaccine.

Antigenicity:

The antigenicity on the same conjugates was analyzed in a sandwich-typeELISA wherein the capture and the detection of antibodies were α-PS andα-PD respectively.

Free Protein Content (%):

The level of “free” residual protein D was determined by using a methodwith SDS treatment of the sample. The conjugate was heated 10 min at100° C. in presence of SDS 0.1% and injected on a SEC-HPLC gelfiltration column (TSK 3000-PWXL). As protein D is dimer, there is arisk of overestimating the level of “free” protein D by dissociation thestructure with SDS.

Molecular Size (K_(av)):

The molecular size was performed on a SEC-HPLC gel filtration column(TSK 5000-PWXL).

Stability:

The stability was measured on a HPLC-SEC gel filtration (TSK 6000-PWXL)for conjugates kept at 4° C. and stored for 7 days at 37° C.

The 11-valent characterization is given in Table 2

The protein conjugates can be adsorbed onto aluminium phosphate andpooled to form the final vaccine.

Conclusion:

Immunogenic conjugates have been produced, that have since been shown tobe components of a promising vaccine. The optimised CDAP conditions forthe best quality final conjugated pneumococcal polysaccharide productwas discovered for each of the 11 valencies. Conjugates of thesepneumococcal polysaccharides obtainable by the above improved(optimised) CDAP process (regardless of the carrier protein, butpreferably protein D) is thus a further aspect of the invention.

Example 2 Study of the Effect of Advanced Adjuvants on theImmunogenicity of the 11-Valent Pneumococcal PS-PD Conjugate Vaccine inInfant Rats

Infant rats were immunised with 11 valent pneumococcal PS-PD conjugatevaccine at a dosage of 0.1 μg each polysaccharide (made according to themethod of Example 1), and using the following adjuvant formulations:none, AlPO₄, 3D-MPL, 3D-MPL on AlPO₄.

The formulation with only 3D-MPL was statistically (and surprisingly)more immunogenic (greatest GMC IgG) than for the other formulations for5 out of 11 antigens. This was true both at high and low concentrationsof 3D-MPL.

Opsonophagocytosis confirmed the GMC results.

Materials and Methods Immunisation Protocol

Infant OFA rats were randomised to different mothers and were 7 days oldwhen they received the first immunisation. They received 2 additionalimmunisations 14 and 28 days later. A bleed was performed on day 56 (28days post III). All vaccines were injected s.c., and there were 10 ratsper vaccine group.

The rats were immunised with an 11 valent pneumococcal conjugate vaccinecomprising the following polysaccharide serotypes conjugated ontoprotein D: 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F.

Formulation

To examine the effect of different advanced adjuvants, the dosage ofconjugate was held constant at 0.1 μg of each polysaccharide, and theadjuvants AlPO₄ and 3D-MPL were formulated in different dosages andcombinations, including no adjuvant at all. These are listed numericallyin Table 3 for reference.

Adsorption on AlPO₄

The concentrated, adsorbed monovalents were prepared according to thefollowing procedure. 50 μg AlPO₄ (pH 5.1) was mixed with 5 μg conjugatedpolysaccharides for 2 hours. The pH was adjusted to pH 5.1 and themixture was left for a further 16 hours. 1500 mM NaCl was added to makeup the salt concentration to 150 mM. After 5 minutes 5 mg/mL2-phenoxyethanol was added. After a further 30 minutes the pH wasadjusted to 6.1, and left for more than 3 days at 4° C.

Preparation of Diluents

Three diluents were prepared in NaCl 150 mM/5 mg/mL phenoxyethanol

A: AlPO₄ at 1 mg/ml.

B: 3D-MPL on AlPO₄ at 250 and 1000 μg/ml respectively Weight ratio3D-MPL/AlPO₄=5/20

C: 3D-MPL on AlPO₄ at 561 and 1000 μg/ml respectively Weight ratio3D-MPL/AlPO₄=50/89

Preparation of Adsorbed Undecavalent

The eleven concentrated, adsorbed PS-PD monovalents were mixed at thecorrect ratio. The complement of AlPO₄ was added as the diluent A. Whenrequired, 3D-MPL was added either as an aqueous solution (non adsorbed,Way 1 see below) or as the diluent B or C (3D-MPL adsorbed on AlPO₄ at 2doses, Way 2, see below).

Way 1

3D-MPL was added to the combined adsorbed conjugates as an aqueoussuspension. It was mixed to the undecavalent for 10 minutes at roomtemperature and stored at 4° C. until administration.

Way 2

3D-MPL was preadsorbed onto AlPO₄ before addition to the combinedadsorbed conjugates (diluent B and C). To prepare 1 ml of diluent, anaqueous suspension of 3D-MPL (250 or 561 μg) was mixed with 1 mg ofAlPO₄ in NaCl 150 mM pH 6.3 for 5 min at room temperature. This solutionwas diluted in NaCl pH. 6.1/phenoxy and incubated overnight at 4° C.

Preparation of Non-Adsorbed Undecavalent

The eleven PS-PD conjugates were mixed and diluted at the right ratio inNaCl 150 mM pH 6.1, phenoxy. When required, 3D-MPL was added as asolution (non adsorbed).

The formulations for all injections were prepared 18 days before thefirst administration.

ELISA

The ELISA was performed to measure rat IgG using the protocol derivedfrom the WHO Workshop on the ELISA procedure for the quantitation of IgGantibody against Streptococcus pneumoniae capsular polysaccharides inhuman serum. In essence, purified capsular polysaccharide is coateddirectly on the microtitre plate. Serum samples are pre-incubated withthe cell-wall polysaccharide common to all pneumococcus (substance C)and which is present in ca. 0.5% in pneumococcal polysaccharidespurified according to disclosure (EP 72513 B1). JacksonImmunoLaboratories Inc. reagents were employed to detect bound murineIgG. The titration curves were referenced to internal standards(monoclonal antibodies) modeled by logistic log equation. Thecalculations were performed using SoftMax Pro software. The maximumabsolute error on these results expected to be within a factor of 2. Therelative error is less than 30%.

Opsonophagocytosis

Opsonic titres were determined for serotypes 3, 6B, 7F, 14, 19F and 23Fusing the CDC protocol (Streptococcus pneumoniae Opsonophagocytosisusing Differentiated HL60 cells, version 1.1) with purified human PMNand baby rabbit complement. Modification included the use of in-housepneumococcal strains, and the phagocytic HL60 cells were replaced bypurified human neutrophils PMN (there, is a high degree of correlationbetween these phagocytic cells). In addition, 3 mm glass beads wereadded to the microtitre wells to increase mixing, and this allowedreduction of the phagocyte:bacteria ratio which was recommended to be400.

Results IgG Concentrations

The geometric mean IgG concentrations determined for every serotype, andPD are shown in Tables 4 to 10. For serotypes 6B, 14, 19F and 23F,previous results obtained using a tetravalent formulation are includedfor comparison.

The highest IgG concentrations have been highlighted in Tables 4 to 10.The statistical p value for 3D-MPL compositions vs. 3D-MPL/AlPO₄compositions is in Table 11. Adjuvant formulation number 4 (non-adsorbedconjugates with high dose 3D-MPL) that gives the highest GMC's for 9 outof 11 cases. In 5/11 cases, MPL at the low dose is the second mostimmunogenic. In addition, adjuvantation gives higher GMC's than bymodifying the dose for all serotypes (data not shown), and this isstatistically significant for serotypes 4, 6B, 7F, 18C and 23F (p<0.05from 95% CI).

Opsonophagocytosis

Opsonophagocytosis results on pooled sera is shown for serotypes 3, 6B,7F, 14, 19F and 23F in Tables 4 to 8. For the most part, these opsonictitres confirm the GMC IgG. Indeed, the correlation with IgGconcentration is greater than 85% for serotypes 6B, 19F, 23F (data notshown). For serotype 3, it is important to note that only the 3D-MPLgroup induced opsonic activity above the threshold.

Conclusions

In this experiment, it was unexpected that the use of 3D-MPL alone wouldinduce the highest IgG concentrations.

The maximal GMC IgG obtained with modifying the adjuvant was comparedwith the maximal GMC obtained by modifying the PS dosage, and it wasfound that 3D-MPL could induce significantly higher responses in 5/11serotypes.

Table 11 shows that when 3D-MPL and 3D-MPL/AlPO₄ compositions arecompared (comparing the process of formulation, and the dose of 3D-MPL),5 of the polysaccharide conjugates are significantly improved, in termsof immunogenicity, when formulated with just 3D-MPL rather than 3D-MPLplus AlPO₄: PS 4, PS 6B, PS 18C, PS 19F, and PS 23F.

Example 3 Study of the Effect of Combination on the Immunogenicity of PS4, PS 6B, PS 18C, PS 19F, and PS 23F Conjugates in Adult Rats

Adult rats were immunised with pneumococcal polysaccharide-protein Dconjugate vaccines either individually, or combined in a multivalentcomposition (either tetra-, penta-, hepta-, or decavalent). Groups of 10rats were immunised twice 28 days apart, and test bleeds were obtainedon day 28 and day 42 (14 days after the 2^(nd) dose).

The sera were tested by ELISA for IgG antibodies to the pneumococcalpolysaccharides. All conjugates induced specific IgG antibodies asmeasured by ELISA. Table 12 shows the effect of combination ofmonovalent PS 6B, PS 18C, PS 19F, and PS 23F protein D conjugates ontheir immunogenicity in adult rats, as measured by IgG concentration at14 days post 2^(nd) dose.

Statistical analysis was performed on all samples to determine ifdifferences in antibody concentration upon combination were significant.The combination of any of serotypes PS 6B, PS 18C, PS 19F, and PS 23Fprotein D conjugates in a multivalent vaccine did not significantlychange their immunogenicity.

TABLE 1 Specific activation/coupling/quenching conditions of PS S.pneumoniae-Protein D conjugates Serotype 3 1 (μfluid.) 4 5 6B 7F PS 2.03.0 2.0 7.5 5.4 3.0 conc. (mg/ml) PS dissolution NaCl NaCl 2M H₂O H₂ONaCl 2M NaCl 2M 2M PD 5.0 5.0 5.0 5.0 5.0 5.0 conc. (mg/ml) InitialPS/PD 1/1 1/1 1/1 1/1 1/1 1/1 Ratio (w/w) CDAP conc.  0.75  0.75  0.75 0.75  0.75  0.75 (mg/mg PS) pH_(a) = pH_(c) = pH_(q) 9.0/9.0/9.09.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 Serotype 9V14 18C 19F 23F PS 2.5 2.5 2.0 4.0 3.3 conc. (mg/ml) PS dissolution NaCl2M NaCl 2M H₂O NaCl 2M NaCl 2M PD 5.0 5.0 5.0 5.0 5.0 conc. (mg/ml)Initial PS/PD 1/0.75 1/0.75 1/1 1/0.5 1/1 Ratio (w/w) CDAP conc.  0.75 0.75  0.75  0.75  0.75 (mg/mg PS) pH_(a) = pH_(c) = pH_(q) 8.5/8.5/9.09.0/9.0/9.0 9.0/9.0/9.0 10/9.5/9.0 9.0/9.0/9.0

TABLE 2 Specifications of the 11 valent pneumococcoal PS-PD vaccine(first numbers of the batch code indicates serotype) Criteria D01PDJ227D03PDJ236 D4PDJ228 D5PDJ235 D6PDJ209 Ratio 1/0.66 1/1.09 1/0.86 1/0.861/0.69 PS/Prot (w/w) Free polysac. 1 1 7 9 0 content (%) <10% Freeprotein 8 <1 19 21 9 content (%) <15% DMAP 0.2 0.6 0.4 1.2 0.3 content(ng/μg PS) <0.5 ng/μg PS Molecular 0.18 0.13 0.12 0.11 0.13 size(K_(av)) Stability no shift no shift no shift low shift no shiftD07PDJ225 D09PDJ222 D14PDJ202 D18PDJ221 D19PDJ206 D23PDJ212 Ratio 1/0.581/0.80 1/0.68 1/0.62 1/0.45 1/0.74 PS/Prot (w/w) Free polysac. 1 <1 <1 44 0 content (%) <10% Free protein 8 0.3 3 21 10 12 content (%) <15% DMAP0.1 0.6 0.3 0.2 0.1 0.9 content (ng/μg PS) <0.5 ng/μg PS Molecular 0.140.14 0.17 0.10 0.12 0.12 size (K_(av)) Stability no shift no shift noshift no shift shift no shift

TABLE 3 Summary Table of Adjuvant Formulations tested with 11-ValentPneumococcal PS-PD in Infant Rats Group AlPO4 MPL Method Description 1None 2 100 AlPO4 3 5 MPL low 4 50 MPL High 5 100 5 Way 1 Way 1 low 6 10050 Way 1 Way 1 high 7 100 5 Way 2 Way 2 low 8 100 50 Way 2 Way 2 high

TABLE 4 Serotype 6B Geometric Mean IgG Concentration, Seroconversion,and Mean Opsonic Titre on Day 28 Post III Immunisation of Infant Ratswith 11-Valent PS-PD using Different Adjuvants (And Comparison withTetravalent Immunisation) 6B 6B GMC 6B 6B GMC 6B 6B IgG Sero- Opso IgGSero- Opso AlPO4 MPL (μg/ml) conversion Titre* (μg/ml) conversion Titre*Group μg μg Method Tetravalent Undecavalent 1 0.047 2/10 12.5 0.004 1/10<6.25 2 100 0.048 4/10 65 0.019 4/10 <6.25 3 5 1.345 10/10  43 4 504.927 10/10  192 5 100 5 1 0.042 7/10 <6.25 6 100 50 1 0.255 10/10 <6.25 7 100 5 2 0.033 3/10 <6.25 0.048 8/10 <6.25 8 100 50 2 0.057 8/10<6.25

TABLE 5 Serotype 14 Geometric Mean IgG Concentration, Seroconversion,and Mean Opsonic Titre on Day 28 Post III Immunisation of Infant Ratswith 11-Valent PS-PD using Different Adjuvants (And Comparison withTetravalent Immunisation) 14 14 GMC 14 14 GMC 14 14 IgG Sero- OpsonicIgG Sero- Opsonic (μg/ml) conversion Titre* (μg/ml) conversion Titre*Group AlPO4 MPL Method Tetravalent Undecavalent 1 0.046  3/10 64 0.0223/10 <6.25 2 100 0.99 10/10 88 0.237 8/10 27 3 5 0.233 10/10  41 4 500.676 10/10  81 5 100 5 1 0.460 9/10 67 6 100 50 1 0.477 10/10  98 7 1005 2 0.81 10/10 49 0.165 8/10 81 8 100 50 2 1.611 10/10  133

TABLE 6 Serotype 19F Geometric Mean IgG Concentration, Seroconversion,and Mean Opsonic Titre on Day 28 Post III Immunisation of Infant Ratswith 11-Valent PS-PD using Different Adjuvants (And Comparison withTetravalent Immunisation) 19F 19F GMC 19F 19F GMC 19F 19F IgG Sero-Opsonic IgG Sero- Opsonic AlPO4 MPL (μg/ml) conversion Titre* (μg/ml)conversion Titre* Group μg μg Method Tetravalent Undecavalent 1 0.042/10 64 0.021  2/10 <6.25 2 100 1.07 9/10 367 0.222  7/10 79 3 5 4.02810/10 296 4 50 21.411 10/10 1276 5 100 5 1 1.649 10/10 172 6 100 50 12.818 10/10 208 7 100 5 2 1.09 10/10  193 0.766 10/10 323 8 100 50 23.539 10/10 241

TABLE 7 Serotype 23F Geometric Mean IgG Concentration, Seroconversion,and Mean Opsonic Titre on Day 28 Post III Immunisation of Infant Ratswith 11-Valent PS-PD using Different Adjuvants (And Comparison withTetravalent Immunisation) 23F 23F GMC 23F 23F GMC 23F 23F IgG Sero-Opsonic IgG Sero- Opsonic AlPO4 MPL (μg/ml) conversion Titre* (μg/ml)conversion Titre* Group μg μg Method Tetravalent Undecavalent 1 0.06 2/10 <6.25 0.152 3/10 <6.25 2 100 0.29 10/10 70 0.56 8/10 <6.25 3 52.296 9/10 389 4 50 4.969 10/10  >1600 5 100 5 1 0.462 5/10 17 6 100 501 0.635 8/10 54 7 100 5 2 0.38 10/10 <6.25 0.203 3/10 18 8 100 50 20.501 7/10 43

TABLE 8 Serotypes 3 and 7F Geometric Mean IgG Concentration,Seroconversion, and Mean Opsonic Titre on Day 28 Post III Immunisationof Infant Rats with 11- Valent PS-PD using Different Adjuvants 3 7F GMC3 3 GMC 7F 7F AlPO4 MPL IgG Sero- Opsonic IgG Sero- Opsonic Group μg μgMethod (μg/ml) conversion Titre* (μg/ml) conversion Titre* 1 0.003  1/10<6.25 0.040 7/10 <6.25 2 100 0.008  6/10 <6.25 0.25 9/10 43 3 5 0.07010/10 <6.25 2.435 10/10  477 4 50 0.108 10/10 18 2.569 10/10  332 5 1005 1 0.015 10/10 <6.25 0.579 10/10  54 6 100 50 1 0.027 10/10 <6.25 0.6119/10 59 7 100 5 2 0.006 10/10 <6.25 0.154 8/10 30 8 100 50 2 0.034 10/10<6.25 0.638 9/10 140

TABLE 9 Serotypes 1, 4 and 5 Geometric Mean IgG Concentration andSeroconversion on Day 28 Post III Immunisation of Infant Rats with11-Valent PS-PD using Different Adjuvants 1 4 5 GMC 1 GMC 4 GMC 5 AlPO4MPL IgG Sero- IgG Sero- IgG Sero- Group μg μg Method (μg/ml) conversion(μg/ml) conversion (μg/ml) conversion 1 0.026  4/10 0.005  0/10 0.040 3/10 2 100 0.282  8/10 0.052  5/10 0.774  9/10 3 5 1.614 10/10 3.45210/10 7.927 10/10 4 50 2.261 10/10 7.102 10/10 13.974 10/10 5 100 5 10.568 10/10 0.676 10/10 3.015 10/10 6 100 50 1 1.430 10/10 0.419  9/105.755 10/10 7 100 5 2 0.478 10/10 0.267  9/10 2.062 10/10 8 100 50 21.458 10/10 0.423 10/10 5.009 10/10

TABLE 10 Serotypes 9V, 18C and PD Geometric Mean IgG Concentration andSeroconversion on Day 28 Post III Immunisation of Infant Rats with11-Valent PS- PD using Different Adjuvants 9V 18C PD GMC 9V GMC 18C GMCPD AlPO4 MPL IgG Sero- IgG Sero- IgG Sero- Group μg μg Method (μg/ml)conversion (μg/ml) conversion (μg/ml) conversion 1 0.018 0/10 0.013 1/10 0.003  0/10 2 100 0.489 6/10 0.092  5/10 0.993 10/10 3 5 0.4827/10 6.560 10/10 3.349 10/10 4 50 11.421 10/10  14.023 10/10 5.446 10/105 100 5 1 2.133 9/10 0.690 10/10 11.407 10/10 6 100 50 1 2.558 10/10 1.771 10/10 1.258 10/10 7 100 5 2 1.536 10/10  0.528 10/10 1.665  8/10 8100 50 2 2.448 9/10 0.980 10/10 5.665 10/10

TABLE 11 The statistical significance (p value) of whether certainpneumococcal polysaccharide conjugates had improved immunogenicity whenformulated with 3D-MPL alone versus with 3D-MPL/AlPO4. 50 μg 3D-MPL v 5μg 3D-MPL vs 3D-MPL/AlPO₄ 3D-MPL/AlPO₄ serotype Way 1 Way 2 Way 1 Way 2 1 0.3 0.05 0.079 0.11  3 0.075 0.01 0.27 0.008  4 0.002 0.0003 0.020.003  5 0.04 0.002 0.1 0.12  6B 0.001 0.0001 0.001 0.0006  7F 0.13 0.150.01 0.005  9V 0.02 0.02 0.1 0.04 14 0.65 0.21 0.3 0.66 18C 0.00080.0002 0.006 0.004 19F 0.0009 0.006 0.21 0.04 23F 0.002 0.0004 0.010.0004 A p value under 0.01 is considered highly significant. Way 1 andWay 2 indicate the method of formulation.

TABLE 12 Geometric Mean IgG concentration (μg/mL) on day 14 post 2^(nd)dose after immunisation of adult rats with 1.0 μg polysaccharide-proteinD conjugate alone or combined in tetravalent, pentavalent, heptavalentor decavalent vaccine. These data are combined from 5 separateexperiments. Serotypes 4 6B 18C 19F 23F Vaccines H T H T T Alone 9.30.11 15 5.2 2.5 Combined 4 0.23 3.7 3.7 2.8 T: combined in tetravalent(T) (PS 6B, 14, 19F, 23F), pentavalent (T plus PS 3), heptavalent (H) (Tplus PS 4, 9V and 18C), and decavalent (H plus PS 1, 5 and 7F)combination vaccines. H: combined in heptavalent (H) (T plus PS 4, 9Vand 18C), and decavalent (H plus PS 1.5 and 7F) combination vaccines.

Example 4 Beneficial Impact of the Addition of Pneumolysin and 3D-MPL onthe Protective Effectiveness of PD-Conjugated 11-Valent PolysaccharideVaccine Against Pneumococcal Lung Colonization in Mice ImmunologicalRead-Outs Elisa Dosage of Pneumolysin-Specific Serum IgG

Maxisorp Nunc immunoplates were coated for 2 hours at 37° C. with 100μl/well of 2 μg/ml recombinant native pneumolysin (PLY) diluted in PBS.Plates were washed 3 times with NaCl 0.9% Tween-20 0.05% buffer. Then,serial 2-fold dilutions (in PBS/Tween-20 0.05%, 100 μl per well) of ananti-PLY serum reference added as a standard curve (starting at 670ng/ml IgG) and serum samples (starting at a 1/10 dilution) wereincubated for 30 minutes at 20° C. under agitation. After washing aspreviously described, peroxydase-conjugated goat anti-mouse IgG(Jackson) diluted 5000× in PBS/Tween-20 0.05% were incubated (100μl/well) for 30 minutes at 20° C. under agitation. After washing, plateswere incubated for 15 min at room temperature with 100 μl/well ofrevelation buffer (OPDA 0.4 mg/ml and H₂O₂ 0.05% in 100 mM pH 4.5citrate buffer). Revelation was stopped by adding 50 μl/well HCl 1N.Optical densities were read at 490 and 620 nm by using EMAX®immunoreader (Molecular Devices). Antibody titre were calculated by the4 parameter mathematical method using SoftMax Pro software.

Hemolysis Inhibition

This assay was done for measuring the ability of serum antibodies toinhibit the pneumolysin (PLY) hemolytic activity. In order to eliminatethe cholesterol (susceptible of interacting with PLY), serum sampleswere treated 2× as follows: they were mixed with 1 equal volume ofchloroform and then incubated for 45 minutes under agitation.Supernatants were collected after centrifugation for 10 minutes at 1000rpm. Cholesterol-cleared sera were diluted (serial 2-fold dilutions in 1mM dithiothreitol, 0.01% BSA, 15 mM TRIS, 150 mM NaCl, pH 7.5) in 96well microplates (Nunc). Fifty μl of a solution containing 4 HU(Hemolysis Unit) of PLY were added in each well and incubated for 15minutes at 37° C. Then, 100 μl of sheep red blood cells (1% solution)were added for 30 minutes at 37° C. After centrifugation for 10 minutesat 1000 rpm, supernatants (150 μl) were collected and put into another96-well microplate for optical density reading at 405 nm. Results wereexpressed as mid-point dilution titers.

Pneumolysin Chemical Detoxification

Recombinant native pneumolysin (PLY) was dialyzed against Phosphate 50mM NaCl 500 mM pH 7.6 buffer. All following steps were done at 39.5° C.under episodic agitation. At day 1, Tween-80 10% (1/250 v/v), N-acetyltryptophan 57.4 mM pH 7.6 (3/100 v/v), glycin 2.2 M in Phosphate buffer(1/100 v/v) and formaldehyde 10% in Phosphate buffer (3/100 v/v) wereadded into PLY solution. At days 2 and 3, formaldehyde 10% was addedagain, at 3/100 and 2/100 v/v ratio, respectively. Incubation at 39.5°C. was sustained until day 7 under episodic agitation. Finally, PLY wasdialyzed against Phosphate 50 mM NaCl 500 mM pH 7.6 buffer. Completeinactivation of PLY was demonstrated in the hemolysis assay.

Pneumococcal Intranasal Challenge in OF1 Mice

Seven week-old OF1 female mice were intranasally inoculated underanesthesia with 5.10⁵ CFU of mouse-adapted S. pneumoniae serotype 6B.Lungs were removed at 6 hours after challenge and homogenized (Ultramax,24000 rpm, 4° C.) in Todd Hewith Broth (THB, Gibco) medium. Serial10-fold dilutions of lung homogenates were plated overnight at 37° C.onto Petri dishes containing yeast extract-supplemented THB agar.Pneumococcal lung infection was determined as the number of CFU/mouse,expressed as logarithmic weighted-average. Detection limit was 2.14 logCFU/mouse.

Example 4A 3D-MPL Adjuvant Effect on Anti Pneumolysin Immune Response

In the present example, we evaluated the impact of 3D-MPL adjuvantationon the immune response to native recombinant pneumolysin (PLY, providedby J. Paton, Children's Hospital, North Adelaide, Australia) and itschemically detoxified counterpart (DPLY). Chemical detoxification wasdone as described above.

Groups of 10 female 6 week-old Balb/c mice were intramuscularlyimmunized at days 0, 14 and 21 with 1 μg PLY or DPLY contained in eitherA: AlPO4 100 μg; or B: AlPO4 100 μg+5 μg 3D-MPL (3 de-O-acylatedmonophosphoryl lipid A, supplied by Ribi Immunochem). FIGS. 1A and 1Bshow ELISA IgG and Hemolysis Inhibition titers (HLI) measured inpost-III sera.

Whichever the antigen, best immune responses were induced in animalsvaccinated with 3D-MPL-supplemented formulations. Interestingly, DPLYwas as immunogenic as PLY when administered with AlPO4+3D-MPL, whilebeing a weaker immunogen in AlPO4 formulation. This showed theadvantageous ability of 3D-MPL to improve the antibody response todetoxified pneumolysin.

In compositions containing pneumolysin, it may be preferable to usechemically detoxified pneumolysin rather than mutationally detoxifiedpneumolysin. This is because detoxified mutants obtained to date stillhave residual toxin activity—chemically detoxifed pneumolysin does not.It is therefore considered another aspect of the invention that, ingeneral, compositions comprising pneumolysin (or pneumolysin mutants)that has been chemically detoxified for use in a vaccine, should beadjuvanted with a Th1 adjuvant, preferably 3D-MPL. Such compositions areprovided by the invention. A method of increasing the immune response ofchemically-detoxifed pneumolysin within an immunogenic compositioncomprising the steps of adding a Th1 adjuvant (preferably 3D-MPL) to thecomposition, is also envisaged.

Example 4B Beneficial Impact of the Addition of an Attenuated Mutant ofPneumolysin and 3D-MPL Adjuvant on the Protective Effectiveness ofPD-Conjugated 11-Valent Polysaccharide Vaccine Against Pneumococcal LungColonization in OF1 Mice Intranasally Challenged with Serotype 6B

In the present example, we evaluated the prophylactic efficacy of avaccine containing the 11-valent polysaccharide-protein D conjugate,attenuated mutant pneumolysin antigen (PdB, WO 90/06951) andAlPO4+3D-MPL adjuvants, compared to the classical AlPO4-adsorbed11-valent polysaccharide-protein D conjugate formulation.

Groups of 12 female 4 week-old OF1 mice were immunized subcutaneously atdays 0 and 14 with formulations containing A: 50 μg is AlPO4; B: 0.1 μgPS/serotype of PD-conjugated 11-valent polysaccharide vaccine+50 μgAlPO4; or C, 0.1 μg PS/serotype of PD-conjugated 11-valentpolysaccharide vaccine+10 μg PdB (provided by J. Paton, Children'sHospital, North Adelaide, Australia)+50 μg AlPO4+5 μg 3D-MPL (suppliedby Ribi Immunochem). Challenge was done at day 21 as described above.

As shown in FIG. 1C, a very significant protection (p<0.007) wasconferred by the 11-valent polysaccharide conjugate vaccine supplementedwith PdB and adjuvanted with AlPO4+MPL (black bars represent thearithmetic mean). On the contrary, no significant protection wasobserved in animals immunized with the 11-valent polysaccharideconjugate/AlPO4 formulation. This result proved that the addition ofpneumolysin antigen (even attenuated) and 3D-MPL adjuvant enhanced theeffectiveness of the 11-valent polysaccharide conjugate vaccine againstpneumonia.

Example 4C Immune Correlates of the Protection Showed in Example 4B

In order to establish the immune correlates of protection conferred inexample 4B, by the 11-valent polysaccharide conjugate vaccinesupplemented with attenuated mutant pneumolysin (PdB) and 3D-MPL,pre-challenge serological antibody responses to polysaccharide 6B andPdB were measured as described above.

Antibody titers were then compared to bacteria colony numbers measuredin lungs of the corresponding animals collected at 6 hourspost-challenge. R² were calculated on Log/Log linear regressions.

Calculated R² were equal to 0.18 and 0.02 for anti-PdD and anti-6Bantibody responses, respectively. This showed the absence of correlationbetween humoral immune responses and protection for both antigens.Anti-6B antibody titers were not significantly different in the groupsimmunized with the 11-valent conjugate vaccine (GMT=0.318 ng/ml) or withthe same vaccine supplemented with PdD and 3D-MPL (GMT=0.458 ng/ml).Therefore, the protection improvement seen with formulation C was notsolely due to a higher antibody response to polysaccharide 6B.

Taken together, the results suggest that protection was not mediated byhumoral immune responses alone, but rather also by a cell-mediatedimmunity induced by the PdB antigen in the presence of 3D-MPL. This gaveadditional support to the addition of protein antigen(s) and potentadjuvant(s) in the pneumococcal polysaccharide conjugate vaccine, so asto coordinate both arms of the immune system for optimal protection.

Example 5 The Cooperation of Both Arms of the Immune System in MiceActively Immunised with Pneumolysin and Passively Immunised withAntibodies Against Pneumococcal PS Example 5A Find the Concentration ofPassively Administered Anti-6B-Polysaccharide (Anti-PS) AntibodyProtecting Against Pneumonia Method

Vaccine Groups: Four groups of 16 mice were passively immunised (i.p.)on day −1 with 100 μl of undiluted rat anti-polysaccharide antiseraaccording to the groups detailed below. (total 64 mice)

IgG Concentration in Group Specificity Antisera G1 α-PS -6B 5 μg/ml. G2α-PS -6B 2 μg/ml. G3 α-PS -6B 0.75 μg/ml.   G4 Control 0 μg/ml.

Animals: 64 male CD-1 mice from Charles River, Canada, weighing approx35 g (approx 10 weeks old).

Anesthesia: Mice were anesthetized with isoflurane (3%) plus O2(1L/min). Organism: S. pneumoniae N1387 (serotype 6) was harvested fromtrypticase soy agar plates (TSA) supplemented with 5% horse blood andsuspended in 6 ml of PBS. Immediately prior to infection, 1 ml bacterialsuspension was diluted into 9 ml of cooled molten nutrient agar (BBL)and kept at 41° C. Mice received approx 6.0 log10 cfu/mouse in a volume50 ul.

Infection: On day 0 mice were anesthetized as described above andinfected with S. pneumoniae N1387 (50 μl cooled bacterial suspension) byintra-bronchial instillation via non-surgical intra-tracheal intubation.This method was described by Woodnut and Berry (Antimicrob. Ag.Chemotherap. 43: 29 (1999)).

Samples: On day 3 post infection, 8 mice/group were sacrificed by CO2overdose and lungs were excised and homogenized in 1 ml PBS. Tenfoldserial dilutions were prepared in PBS to enumerate viable bacterialnumbers. Samples were inoculated (20 μl) in triplicate onto TSA platessupplemented with 5% horse blood and incubated overnight at 37° C. priorto evaluation. Further sets of mice were sacrificed on day 7 and sampledas above.

Results:

IgG conc Bacterial numbers (ug/ml) (log 10 cfu/lungs) at days postinfection in rat sera 3 8 5 6.7 ± 0.7 (1/7) 7.2 ± 0.7 (5/8) 2 6.5 ± 0.7(1/7) 6.9 ± 1.8 (4/7) 0.75 7.7 ± 0.5 (5/8) 4.8 ± 1.4 (2/8) 0 6.7 ± 1.5(3/6) 6.3 ± 1.5 (3/9) Figures in parenthesis are numbers of animals thatdied prior to sample time.Conclusion: In general, there was no significant difference in bacterialnumbers isolated from any of the treatment groups. This indicates thatno measurable protection was afforded by the anti-polysaccharide atconcentrations up to and including 5 μg/ml.

This is similar to what is observed in some human clinical trials, thatis, anti-polysaccharide body is insufficient to protect againstpneumococcal pneumonia in some populations.

Example 5B

Determine the protection from pneumonia afforded by activeadministration of Ply (pneumolysin) with or without adjuvant, andsynergy with sub-optimal anti-PS antibody.

Method

Animals: 128 male CD-1 mice (6 weeks old at old at immunisation, 10weeks old at infection) from Charles River, St. Constant, Quebec,Canada. Animals weighed approx 20 gm at 6 weeks and 38 g at 10 weeks.

Immunisations: Six groups of 16 mice were immunised by subcutaneousinjection on days −22 and −14 with 100 ul of vaccine as detailed below.(Total 128 mice). PdB (WO 90/06951) was obtained courtesy of Dr. JamesPaton, Australia. 3D-MPL was obtained from Ribi/Corixa.

On day −1, specific groups (see Table below) were immunised (i.p. 100μl) passively with a concentration of 4.26 μg/ml (4 ml of 5 μg/ml+1.3 mlof 2 μg/ml) mouse anti-polysaccharide antibody.

Injection Vaccine given Injection Passive Volume days −22, −14 VolumeIgG Group Active (Dosage μg) Passive (day −1) 1-1 100 μl s.c. PdB/AlPO4(10/50) None 1-2 100 μl s.c. PdB/MPL/AlPO4 (10/5/50) None 1-3 100 μls.c. PdB/AlPO4 (10/50) 100 μl i.p. α-PS 1-4 100 μl s.c. PdB/MPL/AlPO4(10/5/50) 100 μl i.p. α-PS 1-5 100 μl s.c. MPL/AlPO4 (5/50) 100 μl i.p.α-PS 1-6 100 μl s.c. MPL/AlPO4 (5/50) None

Infection: On day 0, mice were anesthetized (3% isoflurane plus 1 L/minO2). Bacterial inocula were prepared by harvesting growth of S.pneumoniae N1387 (serotype 6) from trypticase soy agar plates (TSA)supplemented with 5% horse blood and suspending in 6 ml of PBS. Aten-fold dilution (1 ml plus 9 ml) was prepared in cooled moltennutrient agar (kept at 41° C.) immediately prior to infection. Mice wereinfected by intra-bronchial instillation via intra-tracheal intubationand received approximately 6.0 log10 cfu/mouse in a volume of 50 μl.This method was described by Woodnut and Berry (Antimicrob. Ag.Chemotherap. 43: 29 (1999)).

Samples: At 72 post infection, 8 mice/group were sacrificed by CO2overdose and the lungs were excised and homogenized in 1 ml PBS. Tenfoldserial dilutions were prepared in PBS to enumerate viable bacterialnumbers. Samples were inoculated (20 μl) in triplicate onto TSA platessupplemented with 5% horse blood and incubated overnight 37° C. prior toevaluation. Further sets of mice were sacrificed on day 8 post-infectionand samples as above.

Analysis of Data

The outcome measure for comparison of treatment was the number ofbacteria in the lungs at 3 and 7 day post infection. Results arepresented as group means with standard deviations. Statistical analysiswas performed using the Students t-test where a P value of <0.05 wasconsidered significant.

Results: 72 h Post Infection

Bacterial counts from group 1-4 were significantly lower (p<0.05) thanthose from group 1-3.

Bacterial counts from group 1-4 were significantly lower (p<0.05) thanthose from group 1-5.

168 h Post Infection

Bacterial numbers in all groups were approx 2 logs lower at 8 days thanat 3 days, indicating that the infection was resolving.

Bacterial counts from group 1-2 were significantly lower (p<0.05) thanthose from group 1-5.

Day 3 Day 8 Log Standard Log Standard Group CFU/lung Deviation CFU/lungDeviation 1-1 6.93 0.61 5.23 1.28 1-2 6.59 1.25 4.08 1.34 1-3 7.09 0.85.32 1.26 1-4 6.09 1.43 4.46 2.32 1-5 7.19 0.89 5.42 1.05 1-6 6.68 1.145.01 1.48

As demonstrated above, anti-polysaccharide antibody alone (group 1-5)does not afford protection against growth of pneumococci in the lung.PdB adjuvanted with AlPO4 does not confer protection either, but at day8 there is a trend to protection when PdB is combined with 3D-MPL (group1-2).

At Day 3, the group most significantly protected, group 1-4, had allthree elements, PdB, 3D-MPL and passively administeredanti-polysaccharide antibody. This conclusion is supported by themortality rate. Group 1-4 had only 2/8 deaths compared to 5/10 forgroups 1-5 and 1-3.

Conclusion:

As the experiment was done with passively immunised animals, thesynergistic effect of also actively immunising with pneumolysin and MPLcannot be due to an increase in the level of antibodies against thepolysaccharide antigen.

As the animals were only passively immunised against pneumococcalpolysaccharide, by day 8 levels of such antibody would have largelydissipated from the host.

Even so, significant protection against pneumococcal pneumonia could beseen in groups immunised with pneumolysin plus 3D-MPL and especially ingroups immunised with pneumolysin plus 3D-MPL plus passivelyadministered anti-polysaccharide antibody, indicating the synergy ofthis combination.

If the anti-polysaccharide immunisation had been carried out actively(preferably with conjugated polysaccharide), the effect would have beeneven more marked, as the effect of B-cell memory, and constant levels ofanti-PS antibody would have contributed to the immune responsecooperation (see for example FIG. 1C where many of the animals activelyimmunised with polysaccharide and protein was shown to have no bacteriain the lungs after challenge).

Example 6 Immunogenicity in 1-Year-Old Balb/C Mice of 11-ValentPneumococcal-Polysaccharide Protein D Conjugate Vaccine Adjuvanted with3D-MPL

Introduction & objective(s):

Protection against pneumococcal infection is mediated by serotypespecific antibody through opsonophagocytosis. It may be surmised thatincreases in the antibody concentration will result in greaterprotection, and therefore much effort has been expended to find ways toincrease the humoral response. One strategy that has been appliedsuccessfully to conjugate vaccines in pre-clinical studies is the use ofimmunostimulating adjuvants (reviewed in Poolman et al. 1998,Carbohydrate-Based Bacterial Vaccines. In: Handbook of ExperimentalPharmacology eds. P. Perlmann and H. Wigsell. Springer-Verlag,Heidelberg, D).

The data presented in this section show the results of the latestexperiment using clinical lots in a protocol designed to mimic aclinical trial.

Protocol:

One-year-old balb/c mice were immunised with 1/10th of the human dose ofpneumococcal-polysaccharide protein D conjugate vaccine, or 23-valentplain polysaccharide vaccine. The vaccines used were clinical lotsDSP009, DSP013 or DSP014 corresponding to the 1 mcg dosage of serotypes6B and 23F and 5 mcg of the remaining serotypes of the 11-valentconjugated vaccine, the 0.1 mcg dosage of the 11-valent conjugatedvaccine, or the 0.1 mcg dosage of the 11-valent conjugated vaccineadjuvanted with 5 mcg 3D-MPL, respectively. All 11-valent conjugatedvaccines were also adjuvanted with 50 μg AlPO₄.

Groups of 20 mice were immunised intramuscularly. Injections of thegroups listed in the following table were performed on days 0 and 21.Test bleeds were obtained on day 35, (14 days after the second dose).

TABLE Immunisation Schedule for 1-year-old Balb/c mice immunised withclinical lots of pneumococcal- polysaccharide Protein D conjugatevaccine. Day 0 Day 21 Vaccine Vaccine Number Group Dose 1 Dose 2 of mice1 PNEUMOVAX ®-23 Buffer 20 2.5 mcg 2a 11-valent Pn-PD Buffer 20 0.1 mcg2b 11-valent Pn-PD 11-valent Pn-PD 20 0.1 mcg 0.1 mcg 3a 11-valentPn-PD + MPL Buffer 20 0.1 mcg + 5 mcg 3b 11-valent Pn-PD + MPL 11-valentPn-PD + MPL 20 0.1 mcg + 5 mcg 0.1 mcg + 5 mcg 4a 11-valent Pn-PD Buffer20 1/0.5 mcg 4b 11-valent Pn-PD 11-valent Pn-PD 20 1/0.5 mcg 1/0.5 mcgControl Buffer Buffer 20

The sera were tested by ELISA for IgG antibodies to the pneumococcalpolysaccharides following the CDC/WHO consensus protocol, that is, afterneutralisation of the sera with cell-wall polysaccharide. The ELISA wascalibrated to give antibody concentrations in mcg/ml using serotypespecific IgG1 monoclonal antibodies.

Statistical analyses of comparisons were calculated using UNISTAT®version 5.0 beta. ANOVA by the Tukey-HSD method was performed on logtransformed IgG concentrations. Pairwise comparison of seroconversionrates was performed using Fisher's exact test.

Results:

The GMC IgG and 95% confidence interval against the 11 serotypes andprotein D induced 14 days after the second immunisation (dose 2) areshown in the following table. Seroconversion rates are shown where a 95%confidence interval could not be calculated.

Group 1 shows the effect of immunisation with plain polysaccharides,which normally induce only IgM in animals. Most IgG levels are below thethreshold of detection; nevertheless, balb/c mice were able to make IgGto a few pneumococcal polysaccharides, notably serotypes 3, 19F and 14.

Immunisation with conjugate vaccines induced IgG antibody with highseroconversion rates against all serotypes except 23F.

A dosage-dependent response (group 4 vs group 2) was observed only forserotypes 7F and 19F, but these observations were not statisticallysignificant. A greater response was observed after two doses (b groupsvs a groups) for serotypes 3, 6B, 7F and 19F, and PD, and theseobservations were statistically significant in many cases with all 3formulations.

Most interesting is the effect of 3D-MPL. Two doses of the 3D-MPLformulated vaccine (group 3b) induced the highest GMC of specific IgG,and this was statistically significant for all serotypes except 23F, inwhich case it had a significantly higher seroconversion rate (p=0.02group 3b vs 2b, Fisher's exact).

TABLE Geometric Mean [IgG] and 95% Confidence Intervals to SelectedPneumococcal Serotypes and Protein D in 1-Year-Old Balb/c 14 days PostII Immunisation with 11-valent PS-PD Conjugate Vaccine Group 1 2a 2b 3a3b 4a 4b GM [IgG] GM [IgG] GM [IgG] GM [IgG] GM [IgG] GM [IgG] GM [IgG]Sero- μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml type (95% CI) (95% CI)(95% CI) (95% CI) (95% CI) (95% CI) (95% CI)  3 0.24 0.18 0.84 0.72 4.840.22 0.95 (0.16-0.6) (0.11-0.27) (0.47-1.5)  (0.51-1.0)  (3.0-7.9)(0.14-0.35) (0.19-1.8)   6B 0.02 0.04 0.19 0.14 0.74 0.09 0.11 0/20^(#)8/19  (0.09-0.41)  (0.07-0.27)  (0.29-1.9)  (0.05-0.16) (0.05-0.23)  7F0.04 0.07 0.19 0.15 0.97 0.09 0.45 0/20^(#) (0.04-0.12) (0.10-0.39) (0.10-0.22)  (0.49-2.0)  (0.06-0.14) (0.20-1.02) 14 0.15 4.5  6.2  12.9 13.6  4.0  6.9  3/20^(#) (2.5-8.1) (3.6-10.5) (7.8-21.2)  (9.4-19.7)(2.0-8.0)  (4.6-10.6) 19F 1.2  6.7  12.1  10.1  58.5  5.9  22.0 (0.56-2.6)  (3.6-12.5) (7.6-19.3) (5.5-18.5) (42-81) (3.5-9.9)(16.0-30.2) 23F 0.07 0.08 0.08 0.07 0.17 0.06 0.10 1/20^(#) 3/20^(#)2/19^(#) 2/10^(#) 9/20^(#) 1/18^(#) 4/20^(#) PD* 0.25 5.2  11.9  13.5 98.0  10.9  38.7  1/20^(#) (3.3-8.3) (6.9-20.7) (9.5-19.0) (49.1-195.) (6.4-18.4) (21.3-70.3) *In EU/ml; ^(#)Seroconversion rate, defined as 2standard deviations above the average of the negative control. Pleaserefer to previous table for group definitions.

Conclusion:

The data presented here demonstrates that the addition of 3D-MPL to the11-valent pneumococcal-polysaccharide Protein D conjugate vaccineincreased the immune response in elderly balb/c mice to all serotypestested.

In most cases, two doses of vaccine induced higher geometric mean IgGconcentrations that one dose. Since this is not observed using plainpolysaccharide vaccine, even in humans, it is considered an indicationof a T-cell dependent immune response and the induction of immunememory.

These data support a vaccine administation scheme using conjugatedpneumococcal polysaccharides adjuvanted with Th1 adjuvants (preferably3D-MPL), whereby at least two doses of the adjuvanted vaccine areadministered, preferably 1-12 weeks apart, and most preferably 3 weeksapart. Such an administration scheme is considered a further aspect ofthe invention.

The mice used in the experiment were non-responsive to PS 23 (plain orconjugated). Interestingly, although antibody levels against thepolysaccharide remained low regardless of the vaccine composition used,many more mice responded to PS 23 when 3D-MPL was used as the adjuvant(the seroconversion being significantly higher). A use of Th1 adjuvants,particularly 3D-MPL, in vaccine compositions comprising conjugatedpneumococcal polysaccharides in order to relieve non-responsiveness to apneumococcal polysaccharide in a vaccinee is a still further aspect ofthe invention. A method of relieving non-responsiveness with theaforementioned composition using the two dose administration schemedescribed above is yet another aspect.

Example 7 Neisseria Meningitidis C Polysaccharide—Protein D Conjugate(PSC-PD) A: Expression of Protein D

As for Example 1.

B: Manufacture of Polysaccharide C

The source of group C polysaccharide is the strain C11 of N.meningitides. This is fermented using classical fermentation techniques(EP 72513). The dry powder polysaccharides used in the conjugationprocess are identical to MENCEVAX™ (SB Biologicals s.a.).

An aliquot of C11 strain is thawed and 0.1 ml of suspension is streakedon one Mueller Hinton medium petri dish supplemented with yeast extractdialysate (10%, v/v) and incubated for 23 to 25 hrs at 36° C. in a watersaturated air incubator.

The surface growth is then re-suspended in sterilized fermentationmedium and inoculated with this suspension on one Roux bottle containingMueller Hinton medium supplemented with yeast extract dialysate (10%,v/v) and sterile glass beads. After incubation of the Roux bottle during23 to 25 hrs at 36° C. in a water saturated air incubator, the surfacegrowth is re-suspended in 10 ml sterile fermentation medium and 0.2 to0.3 ml of this suspension are inoculated onto 12 other Mueller Hintonmedium Roux bottles.

After incubation during 23 to 25 hrs at 36° C. in a water saturated airincubator, surface growth is re-suspended in 10 ml sterile fermentationmedium. The bacterial suspension is pooled in a conical flask.

This suspension is then aseptically transferred into the fermenter usingsterile syringes.

The fermentation of meningococcus is performed in fermenters containedin a clean room under negative pressure. The fermentation is generallycompleted after 10-12 hrs corresponding to approximately 10¹⁰bacteria/ml (i.e. the early stationary phase) and detected by pHincrease.

At this stage, the entire broth is heat inactivated (12 min at 56° C.)before centrifugation. Before and after inactivation, a sample of thebroth is taken and streaked onto Mueller Hinton medium petri dishes.

C: PS Purification

The purification process is a multi-step procedure performed on theentire fermentation broth. In the first stage of purification, theinactivated culture is clarified by centrifugation and the supernatantis recovered.

Polysaccharide purification is based on precipitation with a quaternaryammonium salt (Cetyltrimethylammonium Bromide/CTAB, CETAVLON®). CTABforms insoluble complexes with polyanions such as polysaccharides,nucleic acid and proteins depending on their pI. Following ioniccontrolled conditions, this method can be used to precipitate impurities(low conductivity) or polysaccharides (high conductivity).

The polysaccharides included in the clarified supernatant areprecipitated using a diatomaceous earth (CELITE® 545) as matrix to avoidformation of insoluble inert mass during the differentprecipitations/purifications.

Purification Scheme for N. meningitidis Polysaccharide C:

Step 1: PSC-CTAB complex re-fixation on CELITE® 545 (diatomaceous earth)and removal of cells debris, nucleic acids and proteins by washing withCTAB 0.05%.

Step 2: Elution of PS with EtOH 50%. The first fractions which areturbid and contain impurities and LPS are discarded. The presence of PSin the following fractions is verified by floculation test.

Step 3: PS-CTAB complex re-fixation on CELITE® 545 (diatomaceous earth)and removal of smaller nucleic acids and proteins by CTAB 0.05% washing.

Step 4: Elution of PS with EtOH 50%. The first turbid fractions arediscarded. The presence of PS in the following fractions is verified byfloculation test.

The eluate is filtered and the filtrate containing crude polysaccharidecollected. The polysaccharide is precipitated from the filtrate byadding ethanol to a final concentration of 80%. The polysaccharide isthen recovered as a white powder, vacuum dried and stored at −20° C.

D: CDAP Conjugation Conjugation of PSC and PD

For conjugation of PSC and PD, the CDAP conjugation technology waspreferred to the classical CNBr activation and coupling via a spacer tothe carrier protein. The polysaccharide is first activated bycyanylation with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate(CDAP). CDAP is a water soluble cyanylating reagent in which theelectrophilicity of the cyano group is increased over that of CNBr,permitting the cyanylation reaction to be performed under relativelymild conditions. After activation, the polysaccharide can be directlycoupled to the carrier protein through its amino groups withoutintroducing any spacer molecule. The unreacted estercyanate groups arequenched by means of extensive reaction with glycine. The total numberof steps involved in the preparation of conjugate vaccines is reducedand most importantly potentially immunogenic spacer molecules are notpresent in the final product.

Activation of polysaccharides with CDAP introduces a cyanate group inthe polysaccharides and dimethylaminopyridine (DMAP) is liberated. Thecyanate group reacts with NH2-groups in the protein during thesubsequent coupling procedure and is converted to a carbamate.

PSC Activation and PSC-PD Coupling

Activation and coupling are performed at +25° C.

120 mg of PS is dissolved for at least 4 h in WFI.

CDAP solution (100 mg/ml freshly prepared in acetonitrile) is added toreach a CDAP/PS (w/w) ratio of 0.75.

After 1 min 30, the pH is raised up to activation pH (pH 10) by additionof triethylamine and is stabilised up to PD addition.

At time 3 min 30, NaCl is added to a final concentration of 2M.

At time 4 min, purified PD is added to reach a PD/PS ratio of 1.5/1; pHis immediately adjusted to coupling pH (pH 10). The solution is left for1 h under pH regulation.

Quenching

6 ml of a 2M glycine solution is added to the PS/PD/CDAP mixture. The pHis adjusted to the quenching pH (pH 8.8). The solution is stirred for 30min at the working temperature, then overnight at +2-8° C. withcontinuous slow stirring.

PS-PD Purification

After filtration (5 μm), the PS-PD conjugate is purified in a cold roomby gel permeation chromatography on a S400HR SEPHACRYL® gel to removesmall molecules (including DMAP) and unconjugated PD: Elution-NaCl 150mM pH 6.5; Monitoring—UV 280 nm, pH and conductivity.

Based on the different molecular size of the reaction components, PS-PDconjugates are eluted first followed by free PD and finally DMAP.Fractions containing conjugate as detected by DMAB (PS) and μBCA(protein) are pooled. The pooled fractions are sterile filtered (0.2 μm)

E: Formulation of PSC-PD adsorbed conjugate vaccine

Washing of AlPO₄

In order to optimize the adsorption of PSC-PD conjugate on AlPO₄, theAlPO₄ is washed to reduce the PO₄ ³ concentration:

-   -   AlPO4 is washed with NaCl 150 mM and centrifuged (4×);    -   the pellet is then resuspended in NaCl 150 mM then filtrated        (100 μm); and    -   the filtrate is heat sterilized.        This washed AlPO₄ is referred to as WAP (washed autoclaved        phosphate).

Formulation Process

The PSC-PD conjugate bulk is adsorbed on AlPO4 WAP before the finalformulation of the finished product. AlPO₄ WAP was stirred with PSC-PDfor 5 minutes at room temperature. The pH was adjusted to 5.1, and themixture was stirred for a further 18 hours at room temperature. NaClsolution was added to 150 mM, and the mixture was stirred for 5 minutesat room temperature. 2-phenoxyethanol was added to 5 mg/mL and themixture was stirred for 15 minutes at room temperature, then adjusted topH 6.1.

Final Composition/Dose

-   -   PSC-PD: 10 μg PS    -   AlPO4 WAP: 0.25 mg Al³⁺    -   NaCl: 150 mM    -   2-phenoxy-ethanol: 2.5 mg    -   Water for Injection: to 0.5 ml    -   pH: 6.1

F: Preclinical Information Immunogenicity of Polysaccharide Conjugate inMice

The immunogenicity of the PSC-PD conjugate has been assessed in 6- to8-weeks-old Balb/C mice. The plain (unadsorbed) conjugate or theconjugate adsorbed onto AlPO4 was injected as a monovalent vaccine.Anti-PSC antibodies induced were measured by ELISA whilst functionalantibodies were analysed using the bactericidal test, both methods beingbased on the CDC (Centers for Disease Control and Prevention, Atlanta,USA) protocols. Results from two different experiments performed toassess the response versus the dose and adjuvant (AlPO₄) effect arepresented.

Dose-Range Experiment

In this experiment, the PSC-PD was injected twice (two weeks apart) inBalb/C mice. Four different doses of conjugate formulated on AlPO4 wereused: 0.1; 0.5; 2.5; and 9.6 μg/animal. The mice (10/group) were bled ondays 14 (14 Post I), 28 (14 Post II) and 42 (28 Post II). Geometric meanconcentrations (GMCs) of polysaccharide C specific antibodies measuredby ELISA were expressed in μg IgG/ml using purified IgG as reference.Bactericidal antibodies were measured on pooled sera and titresexpressed as the reciprocal of the dilution able to kill 50% ofbacteria, using the N. meningitidis C11 strain in presence of babyrabbit complement.

The dose-response obtained shows a plateau from the 2.5 μg dose. Resultsindicate that there is a good booster response between 14 Post I and 14Post II. Antibody levels at 28 Post II are at least equivalent to thoseat 14 Post II. Bactericidal antibody titres are concordant with ELISAconcentrations and confirm the immunogenicity of the PSC-PD conjugate.

Effect of Adjuvant

In this experiment, one lot of PSC-PD conjugate formulated on AlPO4 wasassessed, the plain (non-adjuvanted) conjugate was injected forcomparison. 10 mice/group were injected twice, two weeks apart, by thesubcutaneous route, with 2 μg of conjugate. Mice were bled on days 14(14 Post I), 28 (14 Post II) and 42 (28 Post II), and ELISA andfunctional antibody titres measured (only on 14 Post II and 28 Post IIfor the bactericidal test). The AlPO4 formulation induces up to 10 timeshigher antibody titres as compared to the non-adjuvanted formulations.

Conclusions

The following general conclusions can be made from the results of theexperiments described above:

-   -   PSC-PD conjugate induces an anamnestic response demonstrating        that PSC, when conjugated, becomes a T cell dependent antigen.    -   Anti-PSC antibody concentrations measured by ELISA correlate        well with bactericidal antibody titres showing that antibodies        induced by the PSC-PD conjugate are functional against N.        meningitidis serogroup C.    -   Approximately 2.5 μg of conjugate adsorbed onto AlPO4 appears to        elicit an optimum antibody response in mice.    -   The CDAP chemistry appears to be a suitable method for making        immunogenic PSC-PD conjugates.

Example 8 Preparation of a Polysaccharide from N. meningitidis SerogroupA—PD Conjugate

A dry powder of polysaccharide A (PSA) is dissolved for one hour in NaCl0.2 M solution to a final concentration of 8 mg/ml. pH is then fixed toa value of 6 with either HCl or NaOH and the solution is thermoregulatedat 25° C. 0.75 mg CDAP/mg PSA (a preparation to 100 mg/ml acetonitrile)is added to the PSA solution. After 1.5 minutes without pH regulation,NaOH 0.2M is added to obtain a pH of 10. 2.5 minutes later, protein D(concentrated to 5 mg/ml) is added according to a PD/PSA ratio ofapproximately 1. A pH of 10 is maintained during the coupling reactionperiod of 1 hour. Then, 10 mg glycine (2M pH 9.0)/mg PSA is added and pHregulated at a value of 9.0 for 30 minutes at 25° C. The mixture is thenconserved overnight at 4° C. before purification by exclusion columnchromatography (SEPHACRYL® S400HR from Pharmacia). The conjugate elutesfirst followed by unreacted PD and by-product (DMAP, glycine salts). Theconjugate is collected and sterilized by a 0.2 μm filtration on aSARTOPORE® membrane from Sartorius.

Example 9 In Vitro Characterisations of the Products of Examples 7 and 8

The major characteristics are summarized in the table here below:

Free Conjugate Protein and PS PS/protein Free Protein PS N^(o)description content (μg/ml) ratio (w/w) (%) (%) 1 PS C - PD PD: 2101/0.68 <2 8-9 NaOH for pH PS: 308 regulation 2 PS C - PD PD: 230 1/0.65<2 5-6 TEA for pH PS: 351 regulation 3 PS A - PD PD: 159 1/1.07 5 5-9NaOH for pH PS: 149 regulation

In Vivo Results

Balb/C mice were used as animal model to test the immunogenicity of theconjugates. The conjugates were adsorbed either onto AlPO₄ or Al(OH)₃(10 μg of PS onto 500 μg of Al³⁺) or not adsorbed. The mice wereinjected as followed: 2 injections at two week intervals (2 μgPS/injection).

From these results, we can conclude first that free PS influencesgreatly the immune response. Better results have been obtained withconjugates having less than 10% free PS. The above improvements to theCDAP process is thus a further aspect of the invention.

The formulation is also important. AlPO₄ appears to be the mostappropriate adjuvant in this model. The conjugates induce a boost effectwhich is not observed when polysaccharides are injected alone.

Conclusions

Conjugates of N. meningitidis A and C were obtained with a finalPS/protein ratio of 1 and 0.6-0.7 (w/w) respectively. Free PS and freecarrier protein were below 10% and 15% respectively. Polysacchariderecovery is higher than 70%. Conjugates of PSA and PSC obtainable by theabove improved (optimised) CDAP process (regardless of the carrierprotein, but preferably protein D) is thus a further aspect of theinvention.

Example 10 Preparation of a Polysaccharide from H. influenzae b—PDConjugate

H. influenzae b is one of the major causes of meningitis in childrenunder 2 years old. The capsular polysaccharide of H. influenzae (PRP) asa conjugate onto tetanus toxoid is well known (conjugated by chemistrydeveloped by J. Robbins). CDAP is an improved chemistry. The followingis account of optimal CDAP conditions found for conjugating PRP,preferably to PD.

The parameters influencing the reaction of conjugation are thefollowing:

-   -   The initial concentration of polysaccharide (which can have a        double impact on the final levels of free polysaccharide and on        the sterile filtration step).    -   The initial concentration of the carrier protein.    -   The initial ratio of polysaccharide to protein (which can also        have the double impact on the final levels of free        polysaccharide and on the sterile filtration step).    -   The quantity of CDAP used (usually in large excess).    -   The temperature of the reaction (which can influence the        breakdown of the polysaccharide, the kinetics of the reaction,        and the breakdown of the reactive groups).    -   The pH of activation and coupling.    -   The pH of quenching (influencing the level of residual DMAP).    -   The time of activation, coupling and quenching.

The present inventors have found that the 3 most critical parameters tooptimise the quality of the end product are: the initial ratio ofpolysaccharide/protein; the initial concentration of polysaccharide; andthe coupling pH.

A reaction cube was thus designed with the above 3 conditions as thethree axes. The central points (and experimented value range) for theseaxes were: PS/protein ratio—1/1 (±0.3/1); [PS]=5 mg/ml (±2 mg/ml); andcoupling pH=8.0 (±1.0 pH unit).

The less essential parameters were fixed at the following: 30 mg ofpolysaccharide were used; temperature 25° C.; [CDAP]=0.75 mg/ml PS; pHtitrated with 0.2M NaOH; activation pH=9.5; temperature foractivation=1.5 minutes; coupling temperature—1 hour; [protein]=10 mg/ml;quench pH=9.0; temperature of quenching=1 hour; temperature ofdissolving PS in solvent=1 hour in 2M NaCl; purification on SEPHACRYL®S-400HR eluted with NaCl 150 mM at 12 cm/hour; and filter sterilisingwith a SARTOLAB® P20 at 5 ml/min.

The data looked at to establish optimised conditions when makingproducts within the aforementioned reaction cube were: processdata—maximum yield after filtration, maximum level of proteinincorporated; and quality of product data—final ratio PS/protein, levelof free PS, level of free protein, minimum levels of residual DMAP (abreakdown product of CDAP).

Output from Filtration

The factor which affects the output after filtration is the interactionbetween the initial [PS] and the coupling pH and initial PS/proteinratio. At low [PS] there is little interaction with the latter 2factors, and good filterability always results (approx. 95% for allproducts). However, at high concentrations filterability diminishes ifthe pH and the initial ratio increase (high [PS], lowest ratio, lowestpH=99% filtration; but high [PS], highest ratio and pH=19% filtration).

Level of Incorporation of the Protein

The ratio of the final ratio PS/protein with respect to the initialratio is a measure of the efficiency of coupling. At high [PS], pH doesnot effect the ratio of ratios. However the initial ratio does (1.75 atlow initial ratio, 1.26 at high initial ratios). At low [PS], the ratioof ratios is for the most part lower, however pH now has more of anaffect (low pH, low ratio=0.96; low pH, high ratio=0.8; high pH, lowratio=1.4; and high pH, high ratio=0.92).

Final PS/Protein Ratio

The final ratio depends on the initial ratio and the [PS]. The mostsizeable final ratios are obtained with a combination high initialratios and high [PS]. The effect of pH on the final ratio is not assignificant as a weak [PS].

Level of Free Protein D

The least amounts of free protein D is observed at high pH and high [PS](levels approaching 0.0). The effect of high [PS] becomes especiallymarked when pH is low. The raising of the initial ratio contributes alittle bit to the increase in free protein D.

Residual DMAP

The initial ratio does not have a significant effect. In contrast, thelevel of DMAP increases with the [PS], and decreases when the pH israised.

Conclusions

The most preferable conjugation conditions are thus the following:coupling pH=9.0; [PS]=3 mg/ml; and initial ratio=1/1. With suchconditions the characteristics of the final product are as follows:

Final ratio PS Output from Free protein D DMAP levels PS/proteinfiltration (%) Ratio of ratios (%) (ng/10 μg PS) value range value Rangevalue Range value range value range 1.10 0.91-1.30 92.6 50-138 1.161.03-1.29 0.71 0-10.40 4.95 2.60-7.80

Conjugates of PRP obtainable by the above improved (optimised) CDAPprocess (regardless of the carrier protein, but preferably protein D) isthus a further aspect of the invention.

Example 11 Protein D as an Antigen—how its Protective Efficacy AgainstNon-Typeable H. influenzae can be Improved by Formulating it with 3D-MPL

Female Balb/c Mice (10 per group) were immunized (intramuscularly) withthe eleven valent pneumococcal polysaccharide-protein D conjugatevaccine for a first time at the age of 20 weeks (D0) and received asecond immunization two weeks later (D14). Blood was collected 7 daysafter the second immunization. Antibody titres against protein D weremeasured in terms of the quantity of IgG1, IgG2a and IgG2b typeantibodies.

Freeze-dried undecavalent vaccines (without AlPO₄) were prepared bycombining the conjugates with 15.75% lactose, stirring for 15 minutes atroom temperature, adjusting the pH to 6.1±0.1, and lyophilising (thecycle usually starting at −69° C., gradually adjusting to −24° C. over 3hours, then retaining this temperature for 18 hours, then graduallyadjusting to −16° C. over 1 hour, then retaining this temperature for 6hours, then gradually adjusting to +34° C. over 3 hours, and finallyretaining this temperature over 9 hours).

Composition of formulations and reconstituants for lyophilisates arepresented in Table 13.

The most characteristic measurement as to whether a Th1-type cellmediated immune response has occurred is known to be correlated with thelevel of IgG2a antibody. As can be seen from the data, a surprisinglylarge increase in IgG2a results if the protein D has been lyophilisedwith a Th1 adjuvant (in this case 3D-MPL).

TABLE 13 Composition of formulations (per human dose), and antibodytitres against protein D in mice (with 1/10 dose) Physical PS AlPO₄Caking Pre- IgG1 IgG2a IgG2b IgG1 IgG2a IgG2b state (/500 μl) (/500 μl)Immunostimulanol agent servative Reconstituant μg/ml % liquid 1 μg 500μg  no no 2-PE³ no 76 0.425 0.24 99.1 0.554 0.313 liquid 5 μg 500 μg  nono 2-PE no 66 0.284 0.176 99.3 0.427 0.265 liquid 1 μg 0 μg no no 2-PEno 6.6 0.207 0.036 96.4 3.02 0.526 liquid 5 μg 0 μg no no 2-PE no 5.20.169 0.043 96.1 3.12 0.795 freeze-dried 1 μg 0 μg no lactose no NaCl150 mM¹ 5.2 0.147 0.046 96.4 2.73 0.853 3.15% freeze-dried 5 μg 0 μg nolactose no NaCl 150 mM¹ 11.1 0.11 0.168 97.6 0.967 1.477 3.15%freeze-dried 1 μg 0 μg no lactose no AlPO₄ 45 1.86 0.075 95.9 3.96 0.1603.15% 500 μg² freeze-dried 5 μg 0 μg no lactose no AlPO₄ 19 0.077 0.11999.0 0.401 0.620 3.15% 500 μg² freeze-dried 1 μg 0 μg no lactose no MPL50 μg¹ 45 2.6 3.5 88.1 5.09 6.849 3.15% freeze-dried 5 μg 0 μg nolactose no MPL 50 μg¹ 135 25 5.1 81.8 15.1 3.089 3.15% freeze-dried 1 μg0 μg MPL (50 μg) lactose no buffer¹ 43 22 5.7 60.8 31.1 8.062 3.15%liquid 1 μg 500 μg  MPL (50 μg) no 2-PE no 441 7.1 9.1 96.5 1.55 1.990liquid 5 μg 500 μg  MPL (50 μg) no 2-PE no 299 1.4 0.899 99.2 0.4650.298 ¹before injection; ²+/−2 hours before injection; ³2-phenoxyethanol

1. An immunogenic composition comprising Streptococcus pneumoniaepolysaccharide serotype 18C conjugated to protein D from Haemophilusinfluenzae, and 3-0 deacylated monophosphoryl lipid A (3D-MPL), whereinthe immunogenic composition is substantially devoid of aluminum-basedadjuvant.
 2. The immunogenic composition of claim 1 further comprisingpneumolysin from Streptococcus pneumoniae.
 3. The immunogeniccomposition of claim 2, wherein the pneumolysin is detoxified bychemical treatment or mutation.
 4. A vaccine comprising the immunogeniccomposition of claim
 1. 5. A vaccine comprising the immunogeniccomposition of claim
 2. 6. An immunogenic composition comprisingStreptococcus pneumoniae polysaccharide serotype 18C conjugated toprotein D from Haemophilus influenzae, pneumolysin from Streptococcuspneumoniae, and 3-0 deacylated monophosphoryl lipid A (3D-MPL), whereinthe immunogenic composition is devoid of aluminum-based adjuvant.